Compositions and methods for modulation of effects on phagocyte and lymphoid cell populations employing tirc7

ABSTRACT

Provided are compositions and methods for the prevention and treatment of mammalian disorders that are ameliorated by modulation of effects on phagocyte and lymphoid cell populations and T-cell immune response cDNA 7 (TIRC7) activity in certain cells. Furthermore, improved methods for the production of immunoglobulins to a desired antigen are described. This invention is based on the discovery of a mechanism for the regulation of phagocytosis and the response of lymphoid cell populations to antigens.

FIELD OF THE INVENTION

This invention relates to the prevention and treatment of mammaliandisorders that are ameliorated by modulation of effects on phagocyte andlymphoid cell populations and T-cell immune response cDNA 7 (TIRC7)activity in certain cells. The invention provides numerous compositions,methods and articles of manufacture, and addresses a considerable rangeof disorders such as those of skin and the immune and central nervoussystems. Furthermore, the invention provides improved methods for theproduction of immunoglobulins to a desired antigen. This invention isbased on the discovery of a mechanism for the regulation of phagocytosisand the response of lymphoid cell populations to antigens.

BACKGROUND OF THE INVENTION

There are three main categories of white blood cells, granulocytes,monocytes and lymphocytes. Granulocytes all contain numerous lysosomesand secretory vesicles and are subdivided in neutrophils, eosinophilsand basophils. Monocytes become tissue macrophages, which phagocytoseand digest invading microorganisms and foreign bodies as well as damagedand senescent cells.

Phagocytosis is the cellular process of ingestion, and usually ofisolation or destruction, of particulate material. In vertebrates, it isa characteristic function of various leukocytes and reticuloendothelialcells. Phagocytosis serves as an important bodily defense mechanismagainst infection by microorganisms, and against occlusion of mucoussurfaces and tissues by foreign particles and tissue debris.Phagocytosis is distinct from pinocytosis, which is the uptake of fluidby a cell through invagination and formation of vesicles off the plasmamembrane. Herein, the terms “phagocytosis” and “cellular ingestion” areused interchangeably. The level of phagocytosis in different cells haveimportant implications.

Numerous examples of these implications are provided here:

Immune-Related and Inflammatory Disorders. The primary cause ofpulmonary emphysema is the accumulation of foreign material (e.g. smokecondensate) in the lung. This accumulation is followed by therecruitment of neutrophils that are degranulated during attemptedphagocytosis (Ravis, Am. J. Respir. Crit. Care Med. 150 (1994),5143-5146). Immunological lung disorders such as allergicbronchopulmonary aspergillosis cause mucus plugging of airways,eosinophylic pneumonia and bronchiolitis obliterans. In such diseases,neutrophil elastase cleaved immunoglobulins and digested C3b receptorslimit the phagocytosis of pathogens (Greenberger, JAMA, Vol. 278, No.22, 1997). The increase in neutrophil elastase, while impairingphagocytosis, is beneficial for fighting persistent bacterial infectionsin the lungs, especially in CF patients (Doring, Am. J. Respir. Crit.Care Med. 150: 6 Pt 2, (1994), 114-117).

Periodontal diseases start with the accumulation of plaque at the baseof the teeth, followed by the growth of opportunistic bacteria below thegum line. As with the immune response in emphysema, neutrophils arerecruited to the infected site, followed by their degranulation duringfailed phagocytosis (Travis, Am. J. Respir. Crit. Care Med. Vol. 150(1994), 5143-5146). The rates of adhesion and ingestion of opsonizedStaphylococcus Aureus by polymorphonuclear cells (“PMN's”) fromperiodontal patients is significantly reduced relative to healthycontrols (MacFarlane, J. Periodontol 63 (1992), 908-913).

Individuals who are genetically immuno-compromised, who have acquiredimmuno-suppression (such as HIV infected individuals), or who havetemporarily acquired immuno-suppression (such as that following organtransplantation, foreign implants, valve replacement or cancertreatment, and the like), often suffer from secondary infections.

Pulmonary polymorphnuclear leukocytes from diabetic patients were shownto have reduced phagocytic activities, both at the level of ingestionand killing of bacteria, compared to healthy individuals (e.g. Musclow,Cytobios 65 (1991), medline 15-24). In particular, diabeticabnormalities in the immune response include impaired chemotaxis,impaired phagocytosis and impaired adhesion (Grant-Theule, PeriodontalAbstracts 44 (1996), No. 3). These patients often suffer frominfections.

Cardiovascular System Disorders. The formation of atheroscleroticplaques is induced by aging or by restenosis following balloonangioplasty. Atherosclerotic lesions contain cholesterol-rich particles,many of which aggregate and are internalized in an unregulated fashionby macrophage phagocytosis. This phagocytic process is independent ofthe LDL or scavenger receptor. The lipid-loaded macrophages, calledfoamy cells, can lead to further growth of the atherosclerotic plaque(Hoff, European Heart Journal, II (Supp. E) (1990), 105-115; Robert,Annals New York Acad. of Sciences, 673 (1992), 331-341).

Central Nervous System Disorders. Microglial cells found at theperiphery of amyloid plaque cores have been shown to contain plaquefibrils of beta/A4 amyloid (El Hachimi and Foncin, C. R. Acad. Sci.Paris, Sciences de la vie/Life sciences, 317 (1994), 445-451). Theability of microglial cells to phagocytose and clear senile plaque coresis suppressed in the presence of an astrocyte-secreted diffusablefactor. This factor prevents the clearance of senile plaques, allowingthem to persist in Alzheimer's disease and other neuropathologicaldegenerative processes (DeWitt, Experimental Neurology 149 (1998),329-340). Neutrophil phagocytosis was found to be reduced in clinicallydepressed patients. Patients with phobic disorders have reducedphagocytosis and cell-killing capacities. Benzodiazepine compounds, usedin the treatment of neurological disorders, were shown to reduce orinhibit phagocytosis (e.g. Covelli, Immunopharmacology andImmunotoxicology, 11 (1989),701-714).

Skin Disorders. Mid-dennal elastosis, a skin disorder, is clinicallycharacterized by the appearance of wrinkles and aged appearance whichresults, in part, from phagocytosis of morphologically normal elastictissue (e. g. Fimiani, Arch. Dermatol. Res. 287 (1995), 152-157). Manytypes of pigmentation disorders exist in diverse forms. These can beinherited (e.g. vitiligo), acquired (e.g. post-inflammatory pityriasisalba, idiophatic guttate hypomelanosis, melasma), and transmittedthrough infection (e.g. tinea versicolor). These disorders can be benignand self-limiting (e.g. isolated cafe au lait spots, photocontactdermatitis), or a sign of a more serious underlying disease (e.g.multiple cafe au lait spots, malignant acanthosis nigricans) (Hacker,Postgrad Med. 99 (1996), 177-186). Acne vulgaris is a multi-stagedisorder. The basic acne lesion is the comedo. The second, inflammatorystage when neutrophils are recruited to the comedo area is the reasonthe disease progresses. Nearly all problems associated with acne resultfrom this inflammatory phase.

Furthermore, there are two main classes of lymphocytes, both involved inimmune responses. B lymphocytes make antibodies, while T lymphocyteskill virus-infected cells and regulate the activities of other whiteblood cells. The latter are called helper T cells of which there existtwo types, T_(H)1 cells, which activate macrophages to destroymicroorganisms that they have ingested, and T_(H)2 cells, whichstimulate B cells to proliferate and secrete antibodies. The importanceof phagocytosis in the treatmet of diseases has been discussed before.Besides their role in natural immune response of the body antibodies andantibody producing cells with various immunospecificities are desirablefor therapeutic and diagnostic use, in particular monoclonal antibodies.Antibodies intended for therapeutic and diagnostic use can beproblematic and/or laborious to generate because not every antigen is asuitable immunogen such that, for example, monoclonal antibody producingcells can be obtained. The availability of nonhuman transgenic animals,that are immunogen responsive or adjuvants for use asco-immunostimulatory molecules may make possible the convenientproduction of antibodies against any desired antigen. Furthermore, suchadjuvants may be used in vaccines in order to enhance the immuneresponse in the human body to a foreign antigen.

Hence, there is always a need of alternative and improved means andmethods for regulating cell-mediated immune responses and antibodyresponses.

SUMMARY OF THE INVENTION

This invention provides compositions of matter for treating andpreventing certain mammalian disorders.

These compositions, are based on the discovery of a mechanism for theregulation of phagocytosis and the response of lymphoid cell populationsto antigens.

In a first aspect, the present invention relates to a composition ofmatter for treating: therapeutically or prophylacticly a mammalafflicted with a disorder ameliorated by an increase in phagocytosisand/or monocyte population, which comprises a therapeutically effectiveamount of T-cell immune response cDNA 7 (TIRC7), an activator of TIRC7or of a nucleic acid molecule encoding said TIRC7 or said activator, andoptionally a pharmaceutically or cosmetically acceptable carrier.

In a related aspect, the present invention relates to a composition ofmatter for treating therapeutically or prophylacticly a mammal afflictedwith a disorder ameliorated by a decrease in phagocytosis and/ormonocyte population, which comprises a therapeutically effective amountof an antagonist of T-cell immune response cDNA 7 (TIRC7) or of anucleic acid molecule encoding said antagonist, and optionally apharmaceutically or cosmetically acceptable carrier.

The present invention also relates to a method of increasingphagocytosis and/or monocyte population, i.e. number, comprisingcontacting a mammalian cell with an effective amount of T-cell immuneresponse cDNA 7 (TIRC7), an activator of TIRC7 or of a nucleic acidmolecule encoding said TIRC7 or said activator, and to a method ofdecreasing phagocytosis and/or monocyte population, comprisingcontacting a mammalian cell with an effective amount of an antagonist ofT-cell immune response cDNA 7 (TIRC7) or of a nucleic acid moleculeencoding said antagonist. These methods can be used for treatingtherapeutically or prophylacticly a mammal afflicted with a disorderameliorated by an increase or decrease in phagocytosis and/or monocytepopulation.

The disorders that can be treated in accordance with the methods of theinvention comprise skin disorders, immune system disorders, inflammatorydisorders, respiratory disorders, infectious diseases, diabetes,physical wounds, periodontal disorders and central nervous systemdisorders as well those mentioned in the “background” section.

Furthermore, the present invention relates to an article of manufacturefor administering to a mammal the composition of matter of theinvention, comprising a solid delivery vehicle having the compositionoperably affixed thereto.

In addition, the present invention relates to the use of T-cell immuneresponse cDNA 7 (TIRC7) or a fragment thereof, its encoding orregulatory nucleic acid sequences or anti-TIRC7 antibody for targetingmonocytes, as a target for diagnosis or therapeutic intervention fordiseases related to an increase or decrease in phagocytosis and/orlymphocyte responses, in particular monocyte population in a subject oras a target for screening methods for identifying or isolating agentsfor the treatment of such diseases.

The present invention also concerns a method of diagnosing any one ofthe above mentioned disorders comprising:

-   -   a) assaying a sample from a subject for TIRC7 transcriptional        activity; and    -   b) determining the existence of the disorder characterized by        the induction or suppression of TIRC7 transcriptional activity        compared to a healthy subject,    -   or comprising:    -   a) assaying a sample from a subject for the presence of TIRC7        protein; and    -   b) determining the existence of the disorder by the presence of        TIRC7 protein, wherein the abnormal presence or absence of TIRC7        protein indicates the presence of the disorder.

The present invention also relates to a method of identifying orisolating a therapeutic agent capable of modulating increase or decreasein phagocytosis and/or monocyte population or increasing lymphocyteresponse to antigens in a subject comprising a screening method forantagonists/inhibitors or agonist/activators of TIRC7.

In a second aspect, the present invention relates to a method to producean immunoglobulin or an analog thereof, specific for a desired antigen,which method comprises:

-   -   (a) administering said antigen or an immunogenic portion thereof        to a nonhuman animal under conditions to stimulate an immune        response, whereby said animal produces B cells that secrete        immunoglobulin specific for said antigen; wherein said nonhuman        animal is characterized by being substantially incapable of        producing endogenous T-cell immune response cDNA 7 (TIRC7) or        TIRC7 activity; and    -   (b) recovering said immunoglobulin or analog.

It also relates to the corresponding immortalized B cells, which secreteimmunoglobulin binding to a desired antigen, to their cDNAs and to thecorresponding nonhuman animals as defined above, preferably for use inantibody production.

In a third aspect, the present invention relates to a vaccine useful foreliciting an immune response to a desired antigen comprising atherapeutically effective amount of an antagonist of T-cell immuneresponse cDNA 7 (TIRC7) or of a nucleic acid molecule encoding saidantagonist, optionally further comprising said antigen or immunogenicportion thereof, and optionally further comprising pharmaceuticallyacceptable agents. In this aspect, said antagonist of TIRC7 is used asan adjuvant.

DESCRIPTION OF THE FIGURES

FIG. 1. Generation of TIRC7 deficient mice.

(A) Gene targeting in embryonic stem cells. TIRC7 deficient mice weregenerated by homologous recombination using a vector constructcontaining the neomycin resistance gene which replaced exons 2-8 of theTIRC7 gene.

(B) PCR analysis of genomic DNA from wild type (+/+), heterozygous (±)and homozygous ((−/−)) mice for the disrupted TIRC7 gene locus. PCRprimers were located within the deleted wild type sequence, the neomycincassette and non-deleted 3′ region. PCR revealed a 1.4 kb fragment (wildtype allele) and a 1.2 kb fragment (TIRC7 replaced allele),respectively.

(C) Lack of TIRC7 expression in TIRC7 (−/−) lymphocytes. Flow cytometricanalysis in mouse lymphocytes using a cross-reacting human anti-TIRC7antibody demonstrated significant decrease of TIRC7 expression onheterozygotes (±) and lack of TIRC7 expression on TIRC7 deficient mice((−/−)) in comparison with wild type littermates (+/+).

(D) TIRC7 deficient mice at day 14 (right) displayed about 30% of thebody weight of wild type littermates (left).

(E) Splenocytes isolated from TIRC7 deficient and wildtype (WT) micewere analyzed by flow cytometry to demonstrate lymphocyte subpopulationcounts using anti-CD3 FITC, anti-CD4 PerCP, anti-CD8APC, anti-B220PerCP, anti-CD19 FITC and anti-CD14 conjugated mAbs. Shown is asignificant decrease of all resting T and B cell populations as well asmonocytes in cells lacking TIRC7 in comparison to WT cells. Although Tcell numbers are reduced, no significant changes in CD4/CD8 ratio wasobserved. For monocyte analysis the gate was set on CD14-positive cellsand cell numbers of the WT (188 cells) and TIRC7(−/−) (100 cells)monocyte population are shown in a side scatter vs. CD14 dot plot.

FIG. 2. Histological analysis of TIRC7 (−/−) mice.

(A) Histological staining of TIRC7 (−/−) and WT spleens with hematoxylinand eosin shows a striking hypoplasia of the splenic white pulp ofTIRC7(−/−) (KO) spleens compared to WT littermates. Additionally, TIRC7(−/−) knock out mice show numerous large PALS and small B lymphocyticfollicles in comparison to WT mice.

(B) Immunostaining of TIRC7(−/−) and WT spleens revealed a significanthyperplasia of plasma cells within the splenic red pulpa of TIRC7deficient mice (KO) compared to WT spleens.

FIG. 3. Hyperresponsiveness of T cells from TIRC7 deficient mice.

(A) Proliferation was determined using [³H] thymidine in splenocytesisolated from TIRC7 knock out (KO) and wild type (WT) mice. Cells wereactivated for 48 h with either anti-CD3 mAb alone or in combination withanti-CD28 mAb (a) or PHA (b) at different concentrations. Unstimulated Tcells of wild type (WTo) and TIRC7 deficient mice (KOo) served ascontrols. Compared to wild type cells TIRC7 deficient cells exhibitedhyperreactivity in response to activation stimuli (KO st).

(B) Splenocytes were isolated and either remained non-stimulated (WTo,KOo) or were activated (WTs; KO st) with PHA for 48 h, and culturesupernatants collected after 48 h. IL-2 and IFN-γ cytokine productionwas determined by ELISA in the supernatants of splenocytes from wildtype and TIRC7 (−/−) mice. The results shown are representative of threedifferent wild type and TIRC7 deficient animals, respectively.Significantly elevated cytokine levels are observed in all TIRC7deficient cells.

FIG. 4. Analysis of the Expression Profile of Activation Markers onresting T cells isolated from TIRC7 deficient and Wild-Type Mice.

(A) The expression of CD69 and CD25 as well as CD62L and CD44 wasdetermined in T lymphocytes isolated from wild type (+/+) and TIRC7deficient ((−/−)) mice by flow cytometry analysis with FITC labeledanti-CD3 mAb and PE labeled antibody, respectively. The percentages ofcells positive for the respective marker molecule are indicated in theright upper quadrant of each plot. The gate was set on CD4-positivecells using anti-CD4-PerCP mAb.

(B) Determination of CD11a expression was performed by FACS on restingand activated T lymphocytes, isolated from TIRC7 knock out (−/−) and WT(+/+) mice using FITC-conjugated anti-CD3 mAb and PE-conjugated CD11amAb. Percentages of the naive and memory cell populations are indicatedby boxes in the upper right quadrant of each plot.

(C) The CTLA-4 expression in splenocytes isolated from TIRC7(−/−) andwild type mice was analyzed by FACS. T cells were stained using FITClabeled anti-CD3 mAb combined with PE labeled mouse anti-CTLA-4 mAb. Thegate was set on CD4-positive cells using anti-CD4-PerCP mAb. Shown isthe CTLA-4 expression on the surface of unstimulated cells (a) which isonly minimally increased in TIRC7 deficient mice (−/−) (b) upon 48 h PHAactivation compared with wild type littermates (+/+). Also, insufficientintracellular CTLA-4 expression is observed in TIRC7 deficient micecompared to wild type littermates (c).

(D) CD28 expression was determined by staining splenocytes isolated fromwild type (+/+) and TIRC7 deficient (−/−) mice with anti-CD3-FITClabeled mAb and anti-CD28-PE labeled 20 mAb. ICOS staining was performedusing ICOS Ab followed by staining with secondary goat-anti-mouse-PElabelled Ab.

(E) The expression of CD71 was determined in splenocytes isolated fromwild type (+/+) and TIRC7 deficient (−/−) mice by flow cytometryanalysis gated on CD4 and stained with anti-CD3-FITC labeled mAb andanti-CD71-PE labeled mAb 48 h after activation with PHA. The percentageof cells positive for CD71 is indicated in the right upper quadrant ofeach plot.

FIG. 5. In Vivo T cell response to antigen of TIRC7-Deficient Mice.

(A) Delayed-type hypersensitivity (DTH) response to antigen (ovalbumin)was estimated by measuring foot pad thickness of WT and TIRC7(−/−) mice48 h after re-challenge with ovalbumin. The percentage differences inthe swellings of the foot pads between ova- and PBS-injected controlanimals were estimated. TIRC7(−/−) mice showed a significantly swellingbetween the right and left foot pads compared to WT.

(B) Histology of foot pad-skin obtained from wild type mice (WT) showsexpected mild parenchymal lymphocyte infiltration in dermis (d) whereasTIRC7(−/−) mice (KO) show a severe perivascular and parenchymalinfiltration in stratum reticulare. The sections were isolated after 48h second antigen challenge of DTH response and stained with hematoxylinand eosin ((e) epidermis). Shown is 100× magnification.

FIG. 6. Increased B cell activation in TIRC7 (−/−) mice.

(A) Proliferation of B cells following incubation with various stimuli,i.e. with anti-CD40 antibody alone and with LPS in combination withIL-4, exhibited much higher levels of TIRC7(−/−) B cell (KO st) responsecompared to those of WT littermates (WTst) by Thymidine incorporationassay, respectively. (WTo and KOo represent non stimulated populationsas controls).

(B) Immunoglobulin concentrations in culture supernatants of anti-CD40stimulated splenocytes isolated from WT and TIRC7 deficient mice showsignificant higher levels of IgM and IgG secreted by TIRC7(−/−) B cells(KO st) compared to stimulated WT (WTst) or nonstimulated controls (WTo,KOo). Ig concentrations in the supernatants were determined by ELISAafter 7 days of stimulation.

(C) The activation status of B cells was examined by determining thelevels of various IgGs in the serum of wild type and TIRC (−/−) mice (▴)using ELISA. The results from three different animals, wild type (A) andTIRC7 deficient, respectively, show elevated levels of allimmunoglobulins examined in TIRC7(−/−) mice.

(D) Expression of costimulatory molecule CD86 on B cells after 24 hLPS/IL-4 in vitro stimulation was analyzed by FACS. Staining withanti-B220 PerCP, and anti-CD86 PE conjugated mAb shows that CD86expression is already upregulated in resting status on the surface ofcells from TIRC7 (−/−) deficient mice. Arrows indicate the percentagesof the CD86-high population on activated B cells in the boxes.

FIG. 7. Macrophages revealed extensive morphological and functionaldefects in TIRC7 deficient mice

(A) Macrophages isolated from the peritoneal cavities of TIRC7(−/−) andwild type mice remained non stimulated or were stimulated with LPS andIL-4 for 48 h. TIRC7 deficient mice revealed significant lower numbersof macrophages as can be seen in the unstimulated cell populations.After stimulation, TIRC7(−/−) macrophages showed different morphology ofproliferating macrophages compared to WT littermates.

(B) Confocal microscope images illustrating immunostaining forcytoskeleton proteins such as tubulin (a), vinculin (b) and alpha-actin(c) in macrophages obtained from TIRC7(−/−) and wild type mice (+/+)demonstrated decreased expression of all these proteins in TIRC7deficient cells (−/−).

DETAILED DESCRIPTION OF THE INVENTION

This invention is based on the discovery that TIRC7 deficient miceexhibit increased T and B cell proliferative response to differentstimuli in vitro and in vivo compared to wild type littermates. Theexpression of T cell surface molecules such as CD69 and CD25demonstrated only a moderate increase whereas CD62L and CD44 were foundto be slightly decreased and elevated, respectively, in TIRC7 deficientcells compared to wild type. Strikingly, the expression of costimulatorymolecules such as CTLA4, CD28 and ICOS was significantly reduced whereasno significant changes in expression kinetics was observed for PD1 andCD40L in TIRC7 deficient T cells compared to their littermates. B cellproliferation as well as immunoglobulin expression were induced in TIRC7(−/−) mice splenocytes following activation with IL-4 and LPS.Expression of CD86 was increased in TIRC7 deficient resting B cellswhereas CD80 and CD40 expression remained unchanged. The monocytefraction exhibited a decrease in numbers and failure of phagocytosis andabnormal cytoskeleton architecture. These results demonstrate that TIRC7function is essential for regulating the immune response to variousantigens.

This ability to specifically increase and decrease these cellularfunctions by modulating TIRC7 expression and/or activity permits thetreatment and prevention of disorders, which would be ameliorated by anincrease, or decrease of phagocytosis and/or monocytes. Accordingly,this invention relates a composition of matter for treatingtherapeutically or prophylacticly a mammal afflicted with a disorderameliorated by an increase in phagocytosis and/or monocyte population,which comprises a therapeutically effective amount of T-cell immuneresponse cDNA 7 (TIRC7), an activator of TIRC7 or of a nucleic acidmolecule encoding said TIRC7 or said activator, and optionally apharmaceutically or cosmetically acceptable carrier.

Conclusively, the present invention also relates to a composition ofmatter for treating therapeutically or prophylacticly a mammal afflictedwith a disorder ameliorated by a decrease in phagocytosis and/ormonocyte population, which comprises a therapeutically effective amountof an antagonist of T-cell immune response cDNA 7 (TIRC7) or of anucleic acid molecule encoding said antagonist, and optionally apharmaceutically or cosmetically acceptable carrier.

The term “TIRC7” as used in accordance with the present invention,denotes a protein which initially has been described to be involved inthe signal transduction of T-cell activation and proliferation and that,preferably in a soluble form is capable of inhibiting or suppressingT-cell proliferation in response to alloactivation in a mixed lymphocyteculture or in response to mitogens when exogeneously added to theculture. In vitro translated TIRC7 protein has been shown to be able toefficiently suppress the proliferation of T-cells in a dose dependentmanner in response to alloactivation in a mixed lymphocyte culture or inresponse to mitogens. TIRC7 is known to the person skilled in the artand described, inter alia, in WO99/11782, Utku, Immunity 9 (1998),509-518 and Heinemann, Genomics 57 (1999), 398-406, which also disclosethe amino and nucleic acid sequences of TIRC7.

The agent in the instant compositions that specifically increases ordecreases TIRC7 expression and/or activity can be any type of compoundknown in the art. Examples include, without limitation, organicmolecules, inorganic molecules, peptides, proteins, carbohydrates,nucleic acid molecules, lipids, and any combination thereof. TIRC7antisense nucleic molecules, for example, can be used to decreasephagocytosis. TIRC7 expression vectors or TIRC7 ligands, for example,can be used to increase phagocytosis or monocyte population.

Techniques that can be used for increasing or decreasing the phagocyticactivity of cells in a mammal by modulating TIRC7 activity in accordancewith the present invention can be derived from the prior art. Forexample, in WO95/09011 alternatively to the present invention it isproposed to introduce into appropriate cells a DNA molecule coding foran Fc receptor so that said DNA molecule is expressed and said Fcreceptor thereby produced and the phagocytic activity of said cellsthereby increased. Similarly, but in accordance with the presentinvention TIRC7 encoding DNA would be used. Other approaches that may bemodified and used in accordance with present invention are described forexample in WO96/40199 and WO95/09002.

As used herein, the term “mammal” means any member of the highervertebrate animals included in the class Mammalia, as defined inWebster's Medical Desk Dictionary 407 (1986), and includes but is notlimited to humans, other primates, pigs, dogs, and rodents (such asimmune suppressed mice). In the preferred embodiment of this invention,the mammal is a human.

The instant composition of matter can be of any form known in the art.In one embodiment, the composition comprises a pharmaceuticallyacceptable carrier and one or more discrete pharmaceutical compoundsthat function as the agent that specifically alters TIRC7 expressionand/or activity. In another embodiment, the composition of mattercomprises a naturally-occurring composition, or an extract or componentthereof, which is deemed pharmaceutically or cosmetically acceptable.Such naturally occurring compositions contain certain components whichfunction as active agents, and numerous others that serve aspharmaceutical or cosmetically carriers. The instant compositions can beartificial, naturally occurring, or a combination thereof. In addition,the compositions can be of any physical form known in the art, such asliquids (e. g., solutions, creams, lotions, gels, injectables), solids(e. g., tablets, capsules, powders, granules), aerosols, and coatings.

The terms “antagonist/inhibitor and agonist/activator” in accordancewith the present invention include chemical agents that modulate theaction of TIRC7, either through altering its enzymatic or biologicalactivity or through modulation of expression, e.g., by affectingtranscription or translation. In some cases the antagonist/inhibitor oragonist/activator may also be a substrate or ligand binding molecule.

The term “activator,” as used herein, includes both substances necessaryfor TIRC7 to become active in the first place, and substances whichmerely accentuate its activity.

The term “inhibitor” includes both substances which reduce the activityof the TIRC7 and those which nullify it altogether. When more than onepossible activity is defined herein for TIRC7, the inhibitor oractivator may modulate any or all of TIRC7 activities. An “antagonist”or “agonist” that modulates the activity of TIRC7 and causes for examplea response in a cell based assay refers to a compound that altersdirectly or indirectly the activity of TIRC7 or the amount of activeTIRC7. Typically, the effect of an antagonist is substantially the sameas that of the anti-TIRC7 antibodies described in Utku, Immunity 9(1998), 509-518. Antagonists include competitive as well asnon-competitive antagonists. A competitive antagonist (or competitiveblocker) interacts with or near the site specific for agonist binding. Anon-competitive antagonist or blocker inactivates the function of thereceptor by interacting with a site other than the agonist interactionsite. Preferably, the antagonist/inhibitor and agonist/activator ofTIRC7 are small chemical agents which directly interact with TIRC7.Therefore, there will preferably be a direct relationship between themolar amount of compound required to inhibit or stimulate TIRC7 activityand the molar amount of TIRC7 present or lacking in the cell.

Activators and inhibitors may be designed by structure-assisted computermodeling for example based on alpha-helix and alpha-helix formingregions (“alpha-regions”), beta-sheets and beta-sheet-forming regions(“beta-regions”), turns and turn-forming regions (“turn-regions”), coilsand coil-forming regions (“coil-regions”), hydrophilic regions,hydrophobic regions, alpha amphipathic regions, beta amphipathicregions, flexible regions, surface-forming regions, substrate bindingregion, and high antigenic index regions. Such preferred regions includeGarnier-Robson alpha-regions, beta-regions, turn-regions, andcoil-regions, Chou-Fasman alpha-regions, beta-regions, and turn-regions,Kyte-Doolittle hydrophilic regions and hydrophobic regions, Eisenbergalpha and beta amphipathic regions, Karplus-Schulz flexible regions,Emini surface-forming regions, and Jameson-Wolf high antigenic indexregions. Computer predictions can be made made using for exampleGCG-software derived from HGMP resource center Cambridge (Rice, 1995)

Said agonist/activator of TIRC7 can be or can be derived from, forexample, a TIRC7 polypeptide, a TIRC7 gene, an anti-TIRC7 antibody, atranscription regulator of the TIRC7 gene or a ligand binding molecule,a TIRC7 ligand, or a cell (over)expressing TIRC7. Preferably, said TIRC7polypeptide is a recombinant TIRC7, a functional derivative thereof or afunctionally equivalent substance. DNA sequences encoding TIRC7 as wellas functional derivatives and functionally equivalent substances whichcan be used in the methods and uses of the invention are described inthe prior art; see the references cited above. Moreover, DNA and aminoacid sequences of TIRC7 are available in the Gene Bank database. Asdescribed above, methods for the production of recombinant proteins arewell-known to the person skilled in the art; see, e.g., Sambrook,Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory(1989) N.Y. and Ausubel, Current Protocols in Molecular Biology, GreenPublishing Associates and Wiley Interscience, N.Y. (1989), (1994).

TIRC7 antagonists may be peptides, proteins, nucleic acids, a TIRC7 genetargeting vector, antibodies, small organic compounds, peptide mimics,aptamers or PNAs (Milner, Nature Medicine 1 (1995), 879-880; Hupp, Cell83 (1995), 237-245; Gibbs, Cell 79 (1994), 193-198; Gold, Ann. Rev.Biochem. 64 (1995), 736-797). For the preparation and application ofsuch compounds, the person skilled in the art can use the methods knownin the art, for example those referred to above. Furthermore,antagonists/inhibitors of TIRC7 and methods for obtaining the same aredescribed in, for example, PCT/EP01/12485.

Nucleic acid molecules specifically hybridizing to TIRC7 encoding genesand/or their regulatory sequences may be used for repression ofexpression of said gene, for example due to an antisense or triple helixeffect or they may be used for the construction of appropriate ribozymes(see, e.g., EP-B1 0 291 533, EP-A1 0 321 201, EP-A2 0 360 257) whichspecifically cleave the (pre)-mRNA of a gene encoding TIRC7. The nucleicacid sequence encoding TIRC7 is known in the art; see references supra.Selection of appropriate target sites and corresponding ribozymes can bedone as described for example in Steinecke, Ribozymes, Methods in CellBiology 50, Galbraith et al. eds Academic Press, Inc. (1995), 449-460.Furthermore, methods are described in the literature for identifyingnucleic acid molecules such as an RNA fragment that mimics the structureof a defined or undefined target RNA molecule to which a compound bindsinside of a cell resulting in retardation of cell growth or cell death;see, e.g., WO 98/18947 and references cited therein. These nucleic acidmolecules can be used to identify unknown compounds of pharmaceuticalinterest, and to identify unknown RNA targets for use in treating adisease. Alternatively, for example, the conformational structure of theRNA fragment which mimics the binding site can be employed in rationaldrug design to modify known ligands to make them bind more avidly to thetarget. One such methodology is nuclear magnetic resonance (NMR), whichis useful to identify drug and RNA conformational structures. Stillother methods are, for example, the drug design methods as described inWO 95/35367, U.S. Pat. No. 5,322,933, where the crystal structure of theRNA fragment can be deduced and computer programs are utilized to designnovel binding compounds which can act as antibiotics.

Nucleic acid sequences that are complementary to the TIRC7 encoding genesequence or sense nucleic acid sequences can be synthesized forantisense therapy. These sense or antisense molecules may be DNA, stablederivatives of DNA such as phosphorothioates or methylphosphonates, RNA,stable derivatives of RNA such as 2′-O-alkylRNA, or other TIRC7antisense oligonucleotide mimetics. TIRC7 antisense molecules may beintroduced into cells by microinjection, liposome encapsulation or byexpression from vectors harboring the antisense sequence. TIRC7antisense therapy may be particularly useful for the treatment ofdiseases where it is beneficial to reduce TIRC7 activity. TIRC7 genetherapy may be used to introduce TIRC7 into the cells of targetorganisms. The TIRC7 gene can be ligated into viral vectors that mediatetransfer of the TIRC7 DNA by infection of recipient host cells. Suitableviral vectors include retrovirus, adenovirus, adeno-associated virus,herpes virus, vaccinia virus, polio virus and the like. Alternatively,TIRC7 DNA can be transferred into cells for gene therapy by non-viraltechniques including receptor-mediated targeted DNA transfer usingligand-DNA conjugates or adenovirus-ligand-DNA conjugates, lipofectionmembrane fusion or direct microinjection. These procedures andvariations thereof are suitable for ex vivo as well as in vivo TIRC7gene therapy. TIRC7 gene therapy may be particularly useful for thetreatment of diseases where it is beneficial to elevate TIRC7 activity.Protocols for molecular methodology of gene therapy applicable to theTIRC7 gene are described in Gene Therapy Protocols, edited by Paul D.Robbins, Human press, Totawa N.J., 1996.

Furthermore, the so-called “peptide nucleic acid” (PNA) technique can beused for the inhibition of the expression of a gene encoding a TIRC7.For example, the binding of PNAs to complementary as well as varioussingle stranded RNA and DNA nucleic acid molecules can be systematicallyinvestigated using, e.g., thermal denaturation and BIAcoresurface-interaction techniques (Jensen, Biochemistry 36 (1997),5072-5077). The synthesis of PNAs can be performed according to methodsknown in the art, for example, as described in Koch, J. Pept. Res. 49(1997), 80-88; Finn, Nucleic Acids Research 24 (1996), 3357-3363.Furthermore, folding simulations and computer redesign of structuralmotifs of TIRC7 and its receptors or ligands can be performed asdescribed above to design drugs capable of inhibiting the biologicalactivity of TIRC7.

Preferably, antibodies can be employed in accordance with the presentinvention specifically recognizing TIRC7, or antibody receptors orparts, i.e. specific fragments or epitopes, of such TIRC7s and ligandsthereby inactivating the TIRC7 or the TIRC7 ligand. These antibodies canbe monoclonal antibodies, polyclonal antibodies or synthetic antibodiesas well as fragments of antibodies, such as Fab, Fv or scFv fragmentsetc. Antibodies or fragments thereof can be obtained by using methodswhich are described, e.g., in Harlow and Lane “Antibodies, A LaboratoryManual”, CSH Press, Cold Spring Harbor, 1988 or EP-B1 0 451 216 andreferences cited therein. For example, surface plasmon resonance asemployed in the BIAcore system can be used to increase the bindingefficiency of phage antibodies which bind to an epitope of the TIRC7 orits ligand (Schier, Human Antibodies Hybridomas 7 (1996), 97-105;Malmborg, J. Immunol. Methods 183 (1995), 7-13).

Putative inhibitors which can be used in accordance with the presentinvention including peptides, proteins, nucleic acids, antibodies, smallorganic compounds, ligands, hormones, peptide mimetics, PNAs and thelike capable of inhibiting the biological activity of TIRC7 or itsligand may be identified according to the methods known in the art, forexample as described in EP-A-0 403 506.

In a preferred embodiment of the present invention, the antagonist is anucleic acid molecule and designed to be expressed in monocytes.

In a further preferred embodiment, the antagonist blocks an interactionof TIRC7 and its ligand. Preferably, said antagonist is or comprises

(i) an anti-TIRC7 antibody or an anti-TIRC7-ligand antibody; or

(ii) a non-stimulatory form of TIRC7 or of its ligand.

An anti-TIRC7 antibody to be used in accordance with pharmaceuticalcompositions of the present invention can be preferably a monoclonalantibody, but also include a polyclonal antibody, a single chainantibody, human or humanized antibody, primatized, chimerized or afragment thereof that specifically binds TIRC7 peptide or polypeptidealso including bispecific antibody, synthetic antibody, antibodyfragment, such as Fab, Fv or scFv fragments etc., or a chemicallymodified derivative of any of these. The general methodology forproducing antibodies is well-known and has been described in, forexample, Köhler and Milstein, Nature 256 (1975), 494 and reviewed in J.G. R. Hurrel, ed., “Monoclonal Hybridoma Antibodies: Techniques andApplications”, CRC Press Inc., Boco Raron, Fla. (1982), as well as thattaught by L. T. Mimms et al., Virology 176 (1990), 604-619; see alsoinfra.

Further sources for the basic structure of inhibitors can be employedand comprise, for example, mimetic analogs of the TIRC7 polypeptide.Mimetic analogs of the TIRC7 polypeptide can be generated by, forexample, substituting the amino acids that are expected to be essentialfor the biological activity with, e.g., stereoisomers, i.e. D-aminoacids; see e.g., Tsukida, J. Med. Chem. 40 (1997), 3534-3541.Furthermore, the TIRC7 polypeptide can be used to identify syntheticchemical peptide mimetics that bind to or can function as a ligand,substrate, binding partner or the receptor of the TIRC7 polypeptide aseffectively as does the natural polypeptide; see, e.g., Engleman, J.Clin. Invest. 99 (1997), 2284-2292. For example, folding simulations andcomputer redesign of structural motifs of the protein of the inventioncan be performed using appropriate computer programs (Olszewski,Proteins 25 (1996), 286-299; Hoffman, Comput. Appl. Biosci. 11 (1995),675-679). Computer modelling of protein folding can be used for theconformational and energetic analysis of detailed peptide and proteinmodels (Monge, J. Mol. Biol. 247 (1995), 995-1012; Renouf, Adv. Exp.Med. Biol. 376 (1995), 37-45). In particular, the appropriate programscan be used for the identification of interactive sites of the TIRC7polypeptide and its ligand or other interacting proteins by computerassistant searches for complementary peptide sequences (Fassina,Immunomethods (1994), 114-120. Further appropriate computer systems forthe design of protein and peptides are described in the prior art, forexample in Berry, Biochem. Soc. Trans. 22 (1994), 1033-1036; Wodak, Ann.N. Y. Acad. Sci. 501 (1987), 1-13; Pabo, Biochemistry 25 (1986),5987-5991. Methods for the generation and use of peptide mimeticcombinatorial libraries are described in the prior art, for example inOstresh, Methods in Enzymology 267 (1996), 220-234 and Dorner, Bioorg.Med. Chem. 4 (1996), 709-715. Furthermore, a three-dimensional and/orcrystallographic structure of the TIRC7 protein can be used for thedesign of mimetic inhibitors of the biological activity of the proteinof the invention (Rose, Biochemistry 35 (1996), 12933-12944; Rutenber,Bioorg. Med. Chem. 4 (1996), 1545-1558).

It is also well known to the person skilled in the art, that it ispossible to design, synthesize and evaluate mimetics of small organiccompounds that, for example, can act as a substrate or ligand to theTIRC7 polypeptide. For example, it has been described that D-glucosemimetics of hapalosin exhibited similar efficiency as hapalosin inantagonizing multidrug resistance assistance-associated protein incytotoxicity; see Dinh, J. Med. Chem. 41 (1998), 981-987.

Recombinant TIRC7 polynucleotides, antisense molecules and vectorsincorporating such polynucleotides or antisense molecules can beproduced by methods known to those skilled in molecular biology. Forexample, the choice of vectors which would depend on the functiondesired and include plasmids, cosmids, viruses, bacteriophages and othervectors used conventionally in genetic engineering. Methods which arewell known to those skilled in the art can be used to construct variousplasmids and vectors; see, for example, the techniques described inSambrook, and Ausubel cited supra. Alternatively, the polynucleotidesand vectors can be reconstituted into liposomes for delivery to targetcells. Relevant sequences can be transferred into expression vectorswhere expression of a particular polypeptide is required. Typicalcloning vectors include pBscpt sk, pGEM, pUC9, pBR322 and pGBT9. Typicalexpression vectors include pTRE, pCAL-n-EK, pESP-1, pOP13CAT, pET, pGEX,pMALC, pPIC9, pBac.

The antibodies, nucleic acid molecules, inhibitors and activators usedin the compositions of the present invention preferably have aspecificity at least substantially identical to the binding specificityof the natural ligand or binding partner of the TIRC7 protein, inparticular if TIRC7 stimulation is desired. An antibody or inhibitor canhave a binding affinity to the TIRC7 protein of at least 10⁵ M⁻¹,preferably higher than 10⁷ M⁻¹ and advantageously up to 10¹⁰ M⁻¹ in caseTIRC7 suppression should be mediated.

In a preferred embodiment, a suppressive antibody or inhibitor has anaffinity of at least about 10⁻⁷ M, preferably at least about 10⁻⁹ M andmost preferably at least about 10⁻¹ M; and a TIRC7 stimulating activatorhas an affinity of less than about 10⁻⁷ M, preferably less than about10⁻⁶ M and most preferably in order of 10⁻⁵M.

Disorders that can be treated or prevented using the instant inventioninclude any disorder that can be ameliorated (i.e., a positive effect onthe disorder per se, and/or its secondary effects) by either an increaseor decrease in phagocytosis or monocyte population. These disordersinclude, without limitation, immune system disorders, diabetes,inflammatory disorders, disorders of the central nervous system, skindisorders, physical wounds, periodontal disorders and respiratorydisorders. A number of disorders have characteristics of more than onecategory of disorder. Such disorders include, for example, adhesiondisorders, which can be categorized as both skin disorders and immunesystem disorders. Accordingly, a statement herein that a disorder is ofa particular category (e.g., skin disorder) means that, at the veryleast, the disorder bears traits of that category. Again, however, thedisorder may additionally bear traits of another category. Increasingthe ability of immune cells to ingest foreign objects like bacteria andviruses would be expected to enhance the immune response. For example,mononuclear phagocytes are inactive in chronic microbial infections(Reiner, Immunol. Today 15 (1994), 37481), and their re-activation wouldbe expected to treat the disease. Alternatively, disorders wherein theimmune system is too active would be ameliorated by inhibitingphagocytosis.

Immune system and inflammatory disorders treatable in this inventioninclude, by way of example, AIDS, chemotherapy-induced immunodeficiency,asthma, damage due to toxic substance exposure (e.g., asbestos orsmoke), host rejection of implants and transplanted tissue, adhesiondisorders, mild infections (such as common colds), severe infections(such as meningitis or “killer bacteria”), wounds (such as infected,diabetic, acute and chronic wounds), restenosis, cystic fibrosis,pulmonary emphysema, periodontal disease, and diaper rash. Skindisorders include unwanted pigmentation, unwanted de-pigmentation,psoriasis, rashes, and certain physical skin imperfections (e.g.,wrinkles). In one specific example, vitiligo patients are treated withmelanin (via liposomes or plain) together with a phagocytosis-increasingagent to darken the light spots. Alternatively, they are treated with anagonist of TIRC7 to lighten the darker sites. In an example related toskin disorders, gray hair is treated with melanin (plain orliposome-delivered) and a phagocytosis increasing agent, ideally in ashampoo or cream. Central nervous system disorders include, withoutlimitation, Alzheimer's disease and other senile plaque disorders(treated via up-regulating the phagocytosis of amyloid fibrils),depression, phobic disorders, and other disorders resulting fromsecondary effects of benzodiazepine treatment.

Hence, the present invention provides a method of increasingphagocytosis and/or monocyte population, comprising contacting amammalian cell with an effective amount of T-cell immune response cDNA 7(TIRC7), an activator of TIRC7 or of a nucleic acid molecule encodingsaid TIRC7 or said activator. This method may comprise

-   -   (a) obtaining cells, tissue or an organ from a subject;    -   (b) introducing into said cells, tissue or organ a nucleic acid        molecule encoding and capable of expressing TIRC7 or its ligand        in vivo; and    -   (c) reintroducing the cells, tissue or organ obtained in        step (b) into the same subject or a different subject.

It is envisaged by the present invention that TIRC7 and the nucleic acidmolecules encoding TIRC7 or entities of the corresponding activator areadministered either alone or in combination, and optionally togetherwith a pharmaceutically acceptable carrier or exipient. Said nucleicacid molecules may be stably integrated into the genome of the cell ormay be maintained in a form extrachromosomally, see, e.g., Calos, TrendsGenet. 12 (1996), 463-466. On the other hand, viral vectors described inthe prior art may be used for transfecting certain cells, tissues ororgans. Furthermore, it is possible to use a pharmaceutical compositionof the invention which comprises a nucleic acid molecule encoding aTIRC7 in gene therapy. Suitable gene delivery systems may includeliposomes, receptor-mediated delivery systems, naked DNA, and viralvectors such as herpes viruses, retroviruses, adenoviruses, andadeno-associated viruses, among others. Delivery of nucleic acidmolecules to a specific site in the body for gene therapy may also beaccomplished using a biolistic delivery system, such as that describedby Williams (Proc. Natl. Acad. Sci. USA 88 (1991), 2726-2729).

Standard methods for transfecting cells with nucleic acid molecules arewell known to those skilled in the art of molecular biology, see, e.g.,WO94/29469. Gene therapy to prevent or decrease the development ofdiseases described herein may be carried out by directly administeringthe nucleic acid molecule encoding TIRC7 to a patient or by transfectingcells with said nucleic acid molecule ex vivo and infusing thetransfected cells into the patient.

Furthermore, research pertaining to gene transfer into cells of the germline is one of the fastest growing fields in reproductive biology. Genetherapy, which is based on introducing therapeutic genes into cells byex-vivo or in-vivo techniques is one of the most important applicationsof gene transfer. Suitable vectors and methods for in-vitro or in-vivogene therapy are described in the literature and are known to the personskilled in the art; see, e.g., Giordano, Nature Medicine 2 (1996),534-539; Schaper, Circ. Res. 79 (1996), 911-919; Anderson, Science 256(1992), 808-813; Isner, Lancet 348 (1996), 370-374; Muhlhauser, Circ.Res. 77 (1995), 1077-1086; Wang, Nature Medicine 2 (1996), 714-716;WO94/29469; WO97/00957 or Schaper, Current Opinion in Biotechnology 7(1996), 635-640, and references cited therein. The nucleic acidmolecules comprised in the pharmaceutical composition of the inventionmay be designed for direct introduction or for introduction vialiposomes, or viral vectors (e.g. adenoviral, retroviral) containingsaid nucleic acid molecule into the cell. Preferably, said cell is agerm line cell, embryonic cell, or egg cell or derived therefrom.

Thus, in a preferred embodiment, the nucleic acid molecule comprised inthe pharmaceutical composition for the use of the invention is designedfor the expression of TIRC7 by cells in vivo by, for example, directintroduction of said nucleic acid molecule or introduction of acorresponding plasmid, a plasmid in liposomes, or a viral vector (e.g.adenoviral, retroviral) containing said nucleic acid molecule.

Furthermore, the present invention provides a method to decreasephagocytosis and/or monocyte population, comprising contacting amammalian cell with an effective amount of an antagonist of T-cellimmune response cDNA 7 (TIRC7) or of a nucleic acid molecule encodingsaid antagonist; see supra.

The present invention further provides methods of treatment andprophylaxis regarding mammals affected by a disorder ameliorated by anincrease in phagocytosis and/or monocyte population, which comprisesadministering to the mammal a therapeutically effective amount of T-cellimmune response cDNA 7 (TIRC7), an activator of TIRC7 or of a nucleicacid molecule encoding said TIRC7 or said activator; see supra.

In addition, the present invention provides a method of treating orpreventing in a mammal afflicted with a disorder ameliorated by adecrease in phagocytosis and/or monocyte population, which comprisesadministering to the mammal a therapeutically effective amount of anantagonist of T-cell immune response cDNA 7 (TIRC7) or of a nucleic acidmolecule encoding said antagonist.

Said antagonist or activator for use in the mentioned methods can be anyagent as described above.

The mammalian cells treated in the instant methods are preferably TIRC7expressing cells, and include, without limitation, keratinocytes,fibroblasts, and “professional phagocytes” (i.e., cells havingphagocytosis as a primary function). Professional phagocytes include,for example, neutrophils, macrophages and macrophage-like cells (e.g.,Langerhans cells and Kupfer cells). In the preferred embodiment, themammalian cells are human cells.

In this invention, the “appropriate cells” in which phagocytosis andTIRC7 expression have to be altered in response to the instantcompositions of matter are readily determined based on the nature of thedisorder being treated or prevented. For example, if the disorder beingtreated is a pigmentation disorder, the appropriate cells in which TIRC7expression or activity needs to be altered are keratinocytes.

The instant methods are directed at preventing as well as treatingdisorders. As used herein, “therapeutically treating” a disorder meansreducing the disorder's progression, ceasing the disorder's progression,ceasing or otherwise ameliorating secondary effects of the disorder,reversing the disorder's progression, or preferably, curing thedisorder. As used herein, “prophylactly treating” a disorder meansreducing, and preferably eliminating, the likelihood of the disorder'soccurrence or of occurrence of secondary effects.

In this invention, administering the instant compositions can bealcohols and amino acids, hydrophilic polymers (e.g., polycarbophil andpolyvinylpyrolidone), and adhesives and tackifiers (e.g.,polyisobutylenes, silicone-based adhesives, acrylates and polybutene).Topical delivery of some of the compositions of this invention,particularly those comprising proteins or nucleic acid molecules suchantisense nucleic molecules or TIRC7 expression vectors, can be achievedusing liposomes. The liposomes are preferably nonionic. In one example,they contain (a) glycerol dilaurate; (b) compounds having the steroidbackbone found in cholesterol; and (c) fatty acid ethers having fromabout 12 to about 18 carbon atoms, wherein the constituent compounds ofthe liposomes are in a ratio of about 37.5:12.5:33.3:16.7. Liposomescomprising glycerol dilaurate/cholesterol/polyoxyethylene-10stearylether/polyoxyethylene-9-lauryl ether (“GDL” liposomes) are preferred. Inone embodiment, the liposomes are present in an amount, based upon thetotal volume of the composition, of from about 10 mg/ml to about 100mg/ml, and preferably from about 15 mg/ml to about 50 mg/ml. A ratio ofabout 37.5:12.5:33.3:16.7 is preferred. Methods of preparing liposomesare well known in the art, such as those disclosed in Niemiec, Pharm.Res. 12 (1995), 1184-1188. Also, for topical or transdermaladministration, the instant compositions can be combined with othercomponents such as moisturizers, cosmetic adjuvants, anti-oxidants,bleaching agents, tyrosinase inhibitors and other known depigmentationagents, alpha-hydroxy acids, surfactants, foaming agents, conditioners,humectants, fragrances, viscosifiers, buffering agents, preservatives,sunscreens and the like. The compositions of this invention can alsocontain active amounts of retinoids including, for example, tretinoin,retinol, esters of tretinoin and/or retinol and the like.

Transmucosal delivery systems include patches, tablets, suppositories,pessaries, gels and creams, and can contain excipients such assolubilizers and enhancers (e.g., propylene glycol, bile salts and aminoacids), and other vehicles (e.g., polyethylene glycol, fatty acid estersand derivatives, and hydrophilic polymers such ashydroxypropylmethylcellulose and hyaluronic acid).

Injectable drug delivery systems include solutions, suspensions, gels,microspheres and polymeric injectables, and can comprise excipients suchas solubility-altering agents (e.g., ethanol, propylene glycol andsucrose) and polymers (e.g., polycaprylactones and PLGA's). Systems forcentral nervous system delivery include, for example, a lipidcoupledderivative to cross the blood brain barrier (e.g. DHA). Implantablesystems include rods and discs, and can contain excipients such as PLGAand polycaprylactone.

Oral delivery systems include tablets and capsules. These can containexcipients such as binders (e.g., hydroxypropylmethylcellulose,polyvinyl pyrilodone, other cellulosic materials and starch), diluents(e.g., lactose and other sugars, starch, dicalcium phosphate andcellulosic materials), disintegrating agents (e.g., starch polymers andcellulosic materials) and lubricating agents (e.g., stearates and talc).Such delivery systems also include, for example, toothpaste, mouthwash,lozenges and lollipops.

Solutions, suspensions and powders for reconstitutable delivery systemsinclude vehicles such as suspending agents (e.g., gums, zanthans,cellulosics and sugars), humectants (e.g., sorbitol), solubilizers(e.g., ethanol, water, PEG and propylene glycol), surfactants (e.g.,sodium lauryl sulfate, Spans, Tweens, and cetyl pyridine), preservativesand antioxidants (e.g., parabens, vitamins E and C, ascorbic acid, andnatural extracts), anti-caking agents, coating agents, and chelatingagents (e.g., EDTA). Oil-in-water emulsions, water-in-oil emulsions,solvent-based formulations and aqueous gels known to those of skill inthe art can also be utilized as vehicles for the delivery of thecompositions of this invention.

This invention still further provides an article of manufacture foradministering to a mammal the instant composition of matter, comprisinga solid delivery vehicle having the composition operably (i.e.,deliverably) affixed thereto. The solid delivery vehicle can be anydevice designed to come into temporary or permanent contact with thebody, whether or not it was originally intended for use as a deliveryvehicle. Examples of the instant article of manufacture include, withoutlimitation, coated bandages or other wound dressing for treating wounds,coated bodily implants (including implants with coated internalscaffolding) for either preventing or promoting tissue growth, andcoated balloon catheters and stents for preventing restenosis.

In addition, this invention provides a method of administering atherapeutic; prophylactic or cosmetic compound to a mammal, comprisingadministering to the mammal (a) the compound and (b) a composition ofmatter of the invention comprising a pharmaceutical or cosmetic carrierand an agent that specifically modulates TIRC7 expression and/oractivity in an amount sufficient to increase phagocytosis in cells whereuptake of the compound is desired, wherein the composition isadministered prior to and/or concurrently with the administration of thecompound. The pharmaceutical compound can be, for example, apolypeptide, protein, or nucleic acid molecule. In one embodiment, thepharmaceutical compound and composition are administered together viamicroscopic porous biodegradable beads, which then release thepharmaceutical compound after being ingested through phagocytosis by theappropriate cells.

In accordance with the above, the present invention also relates to theuse of T-cell immune response cDNA 7 (TIRC7) or a fragment thereof, itsencoding or regulatory nucleic acid sequences or anti-TIRC7 antibody fortargeting monocytes, as a target for diagnosis or. therapeuticintervention for diseases related to an increase or decrease inphagocytosis and/or monocyte population in a subject or as a target forscreening methods for identifying or isolating agents for the treatmentof such diseases.

Pharmaceutically useful compositions such as described herein-before,comprising TIRC7 DNA, TIRC7 RNA, or TIRC7 protein, or modulators ofTIRC7 activity, i.e. activator/agonist or inhibitor/antagonist, orchemical derivatives thereof may be formulated according to knownmethods such as by the admixture of a pharmaceutically acceptablecarrier. Examples of such carriers and methods of formulation may befound in Remington's Pharmaceutical Sciences. To form a pharmaceuticallyacceptable composition suitable for effective administration, suchcompositions will contain an effective amount of the protein, DNA, RNA,or modulator. Therapeutic or diagnostic compositions of the inventionare administered to an individual in amounts sufficient to treat ordiagnose disorders in which modulation of TIRC7-related activity isindicated. The effective amount may vary according to a variety offactors such as the individual's condition, weight, sex and age. Otherfactors include the mode of administration. The pharmaceuticalcompositions may be provided to the individual by a variety of routessuch as by intracoronary, intraperitoneal, subcutaneous, intravenous,transdermal, intrasynovial, intramuscular or oral routes.

The term “chemical derivative” describes a molecule that containsadditional chemical moieties that are not normally a part of the basemolecule. Such moieties may improve the solubility, half-life,absorption, etc. of the base molecule. Alternatively the moieties mayattenuate undesirable side effects of the base molecule or decrease thetoxicity of the base molecule. Examples of such moieties are describedin a variety of texts, such as Remington's Pharmaceutical Sciences.

TIRC7 DNA, TIRC7 RNA, or TIRC7 protein, or modulators of TIRC7 activitydisclosed herein may be used alone at appropriate dosages defined byroutine testing in order to obtain optimal activation or inhibition ofthe TIRC7 activity while minimizing any potential toxicity. In addition,co-administration or sequential administration of other agents may bedesirable.

A therapeutically effective dose refers to that amount of protein,antibodies, nucleic acid, agonists, activators, antagonists, orinhibitors which ameliorate the symptoms or condition. Therapeuticefficacy and toxicity of such compounds can be determined by standardpharmaceutical procedures in cell cultures or experimental animals,e.g., ED50 (the dose therapeutically effective in 50% of the population)and LD50 (the dose lethal to 50% of the population). The dose ratiobetween therapeutic and toxic effects is the therapeutic index, and itcan be expressed as the ratio, LD50/ED50.

In a further embodiment the present invention relates to a method ofdiagnosing a disorder related to an increase or decrease in phagocytosisand/or monocyte population in a subject comprising:

-   -   a) assaying a sample from a subject for TIRC7 transcriptional        activity; and    -   b) determining the existence of the disorder characterized by        the induction or suppression of TIRC7 transcriptional activity        compared to a healthy subject.

In a still further embodiment the present invention relates to a methodof diagnosing a disorder related to an increase or decrease inphagocytosis and/or monocyte population in a subject comprising:

-   -   a) assaying a sample from a subject for the presence of TIRC7        protein; and    -   b) determining the existence of the disorder by the presence of        TIRC7 protein, wherein the abnormal presence or absence of TIRC7        protein indicates the presence of the disorder.

Preferably, in said methods the cells to be analyzed are or comprisemonocytes.

In these embodiments, the TIRC7 polynucleotides, nucleic acid molecules,(poly)peptide, antibodies or ligands preferably labeled with adetectable moiety. A variety of techniques are available for labelingbiomolecules, are well known to the person skilled in the art and areconsidered to be within the scope of the present invention. Suchtechniques are, e.g., described in Tijssen, “Practice and theory ofenzyme immuno assays”, Burden, R H and von Knippenburg (Eds), Volume 15(1985), “Basic methods in molecular biology”; Davis L G, Dibmer M D;Battey Elsevier (1990), Mayer et al., (Eds) “Immunochemical methods incell and molecular biology” Academic Press, London (1987), or in theseries “Methods in Enzymology”, Academic Press, Inc. There are manydifferent labels and methods of labeling known to those of ordinaryskill in the art. Commonly used labels comprise, inter alia,fluorochromes (like fluorescein, rhodamine, Texas Red, etc.), enzymes(like horse radish peroxidase, β-galactosidase, alkaline phosphatase),radioactive isotopes (like ³²P or ¹²⁵I), biotin, digoxygenin, colloidalmetals, chemi- or bioluminescent compounds (like dioxetanes, luminol oracridiniums). Labeling procedures, like covalent coupling of enzymes orbiotinyl groups, iodinations, phosphorylations, biotinylations, randompriming, nick-translations, tailing (using terminal transferases) arewell known in the art. Detection methods comprise, but are not limitedto, autoradiography, fluorescence microscopy, direct and indirectenzymatic reactions, etc.

In addition, the above-described compounds etc. may be attached to asolid phase. Solid phases are known to those in the art and may comprisepolystyrene beads, latex beads, magnetic beads, colloid metal particles,glass and/or silicon chips and surfaces, nitrocellulose strips,membranes, sheets, animal red blood cells, or red blood cell ghosts,duracytes and the walls of wells of a reaction tray, plastic tubes orother test tubes. Suitable methods of immobilizing TIRC7 nucleic acids,(poly)peptides, proteins, antibodies, etc. on solid phases include butare not limited to ionic, hydrophobic, covalent interactions and thelike. The solid phase can retain one or more additional receptor(s)which has/have the ability to attract and immobilize the region asdefined above. This receptor can comprise a charged substance that isoppositely charged with respect to the reagent itself or to a chargedsubstance conjugated to the capture reagent or the receptor can be anyspecific binding partner which is immobilized upon (attached to) thesolid phase and which is able to immobilize the reagent as definedabove.

Commonly used detection assays can comprise radioisotopic ornon-radioisotopic methods. These comprise, inter alia, RIA(Radioisotopic Assay) and IRMA (Immune Radioimmunometric Assay), EIA(Enzym Immuno Assay), ELISA (Enzyme Linked Immuno Assay), FIA(Fluorescent Immuno Assay), and CLIA (Chemioluminescent Immune Assay).Other detection methods that are used in the art are those that do notutilize tracer molecules. One prototype of these methods is theagglutination assay, based on the property of a given molecule to bridgeat least two particles.

For diagnosis and quantification of (poly)peptides, polynucleotides,etc. in clinical and/or scientific specimens, a variety of immunologicalmethods, as described above as well as molecular biological methods,like nucleic acid hybridization assays, PCR assays or DNA EnzymeImmunoassays (Mantero et al., Clinical Chemistry 37 (1991), 422-429)have been developed and are well known in the art. In this context, itshould be noted that the TIRC7 nucleic acid molecules may also comprisePNAs, modified DNA analogs containing amide backbone linkages. Such PNAsare useful, inter alia, as probes for DNA/RNA hybridization.

The above-described compositions may be used for methods for detectingexpression of a TIRC7 polynucleotide by detecting the presence of mRNAcoding for a TIRC7 (poly)peptide which comprises, for example, obtainingmRNA from cells of a subject and contacting the mRNA so obtained with aprobe/primer comprising a nucleic acid molecule capable of specificallyhybridizing with a TIRC7 polynucleotide under suitable hybridizationconditions, and detecting the presence of mRNA hybridized to theprobe/primer. Further diagnostic methods leading to the detection ofnucleic acid molecules in a sample comprise, e.g., polymerase chainreaction (PCR), ligase chain reaction (LCR), Southern blotting incombination with nucleic acid hybridization, comparative genomehybridization (CGH) or representative difference analysis (RDA). Thesemethods for assaying for the presence of nucleic acid molecules areknown in the art and can be carried out without any undueexperimentation.

The present invention also relates to a kit for use in any one of theabove described methods, said kit comprising an anti-TIRC7 antibody orTIRC7 antisense nucleic acid molecule, or a derivative thereof Such kitsare used to detect DNA which hybridizes to TIRC7 DNA or to detect thepresence of TIRC7 protein or peptide fragments in a sample. Suchcharacterization is useful for a variety of purposes including but notlimited to forensic analyses, diagnostic applications, andepidemiological studies in accordance with the above-described methodsof the present invention. The recombinant TIRC7 proteins, DNA molecules,RNA molecules and antibodies lend themselves to the formulation of kitssuitable for the detection and typing of TIRC7. Such a kit wouldtypically comprise a compartmentalized carrier suitable to hold in closeconfinement at least one container. The carrier would further comprisereagents such as recombinant TIRC7 protein or anti-TIRC7 antibodiessuitable for detecting TIRC7. The carrier may also contain a means fordetection such as labeled antigen or enzyme substrates or the like.

In addition, the present invention also relates to a method ofidentifying or isolating a therapeutic agent capable of modulatingincrease or decrease in phagocytosis and/or monocyte population orincreasing lymphocyte response to antigens in a subject comprising ascreening method for antagonists/inhibitors or agonist/activators ofTIRC7. Generally, screening methods for antagonists/inhibitors oragonist/activators of TIRC7 are described in WO99/11782 and inPCT/EP01/12485.

Preferably, any one of the above described diagnostic methods, screeningmethods and kits are used in the detection or screening of disordersrelated to phagocytosis and/or lymphocyte activity, most preferablythose described above.

In a further aspect, the present invention relates to a method toproduce an immunoglobulin or an analog thereof, specific for a desiredantigen, which comprises:

-   -   (a) administering said antigen or an immunogenic portion thereof        to a nonhuman animal under conditions to stimulate an immune        response, whereby said animal produces B cells that secrete        immunoglobulin specific for said antigen; wherein said nonhuman        animal is characterized by being substantially incapable of        producing endogenous T-cell immune response cDNA 7 (TIRC7) or        TIRC7 activity in lymphocytes; and    -   (b) recovering said immunoglobulin or analog.

This aspect of the invention is based on the surprising finding that Bcell proliferation as well as immunoglobulin expression were induced inTIRC7 (−/−) mice splenocytes following activation with IL-4 and LPS; seeexamples 6 and 7. Thus, nonhuman animals wherein the activity of TIRC7has been substantially reduced in the appropriate cells, preferably atleast in B cells, for example by knock out or antisense approaches canadvantageously be used for antibody production. Alternatively, thenormal immunization process is accompanied by administering anantagonist/inhibitor of TIRC7 in order to exogenously bring about thesame effect as observed with the TIRC7 (−/−) mice in the examples.Hence, in principle any known method for the production of monoclonalantibodies may be used except that in addition or alternatively TIRC7activity is substantially reduced in at least some if not all of thecells of the nonhuman animal which has been immunized with a desiredantigen. Preferably, TIRC7 activity is substantially reduced in at leastthe lymphocytes of the nonhuman animal, at least at some stage of theimmunization process. For production of the desired antibodies, thefirst step is administration of the antigen. Techniques for suchadministration are conventional and involve suitable immunizationprotocols and formulations which will depend on the nature of theantigen per se. It may be necessary to provide the antigen with acarrier to enhance its immunogenicity and/or to include formulationswhich contain adjuvants and/or to administer multiple injections and/orto vary the route of the immunization, and the like. Such techniques arestandard and optimization of them will depend on the characteristics ofthe particular antigen for which immunospecific reagents are desired.Such methods including methods of immunization to enhance the immuneresponse to specific antigens in vivo are well known in the art and aredescribed for example in Rudbach, Methods Mol. Biol. 45 (1995), 1-8 andDean, Methods Mol. Biol. 80 (1998), 23-37. The method of the presentinvention also encompasses methods to produce human antibodies such asdescribed in WO96/33735 with the mentioned modifications. As mentionedbefore, the effect of reducing TIRC7 activity may be achieved by meansother that inactivating the TIRC7 gene. Thus, in one embodiment theantigen or an immunogenic portion thereof is administered in conjunctionwith an TIRC7 antagonist as described in the afore mentioned embodimentsto the nonhuman animal.

As used herein, the term “immunospecific reagents” includesimmunoglobulins and their analogs. The term “analogs” has a specificmeaning in this context. It refers to moieties that contain theimmunoglobulin which account for its immunospecificity. In particular,complementarity determining regions (CDRs) are required, along withsufficient portions of the framework (FRs) to result in the appropriatethree dimensional conformation. The person skilled in the art knows thateach variable domain (the heavy chain V_(H) and light chain V_(L)) Of anantibody comprises three hypervariable regions, sometimes calledcomplementarity determining regions or “CDRs” flanked by four relativelyconserved framework regions or “FRs”. The CDRs contained in the variableregions of the antibody of the invention can be determined, e.g.,according to Kabat, Sequences of Proteins of Immunological Interest(U.S. Department of Health and Human Services, third edition, 1983,fourth edition, 1987, fifth edition 1990 and updated ones). Typicalimmunospecific analogs of antibodies include F(ab″)2, Fab′, and Fabregions. Modified forms of the variable regions to obtain, for example,single chain Fv analogs with the appropriate immunospecificity areknown. A review of such Fv construction is found, for example, inHuston, Methods in Enzvmology 203 (1991), 46-63. The construction ofantibody analogs with multiple immunospecificities is also possible bycoupling the variable regions from one antibody to those of secondantibody.

The variable regions can also be coupled to a variety of additionalsubstances which can provide toxicity, biological functionality,alternative binding specificities and the like. The moieties includingthe variable regions produced by the methods of the invention includesingle-chain fusion proteins, molecules coupled by covalent methodsother than those involving peptide linkages, and aggregated molecules.Examples of analogs which include variable regions coupled to additionalmolecules covalently or noncovalently include those in the followingnonlimiting illustrative list. Traunecker, Int. J. Cancer Surp. SuDP 7(1992), 51-52, describe the bispecific reagent janusin in which the Fvregion directed to CD3 is coupled to soluble CD4 or to other ligandssuch as OVCA and IL-7. Similarly, the variable regions produced by themethod of the invention can be constructed into Fv molecules and coupledto alternative ligands such as those illustrated in the cited article.Higgins, J. Infect Disease 166 (1992), 198-202, described aheteroconjugate antibody composed of OKT3 cross-linked to an antibodydirected to a specific sequence in the V3 region of GP120. Suchheteroconjugate antibodies can also be constructed using at least thevariable regions contained in the immunoglobulins produced by theinvention methods. Additional examples of specific antibodies includethose described by Fanger, Cancer Treat. Res. 68 (1993), 181-194 and byFanger, Crit. Rev. Immunol. 12 (1992), 101-124. Conjugates that areimmunotoxins including conventional antibodies have been widelydescribed in the art. The toxins may be coupled to the antibodies byconventional coupling techniques or immunotoxins containing proteintoxin portions can be produced as fusion proteins. The analogs of thepresent invention can be used in a corresponding way to obtain suchimmunotoxins. Illustrative of such immunotoxins are those described byByers, Seminars Cell. Biol. 2 (1991), 59-70 and by Fanger, Immunol.Today 12 (1991), 51-54.

It will also be noted that some of the immunoglobulins and analogs ofthe invention will have agonist activity with respect to antigens forwhich they are immunospecific in the cases wherein the antigens performsignal transducing functions. Thus, a subset of antibodies or analogsprepared according to the methods of the invention which areimmunospecific for, for example, a cell surface receptor, will becapable of eliciting a response from cells bearing this receptorcorresponding to that elicited by the native ligand. Furthermore,antibodies or analogs which are immunospecific for substances mimickingtransition states of chemical reactions will have catalytic activity.Hence, a subset of the antibodies and analogs of the invention willfunction as catalytic antibodies.

Naturally, the method of the present invention can further compriserecovering said polyclonal immunoglobulin or analog from said animal.Furthermore, the method of the invention may further compriseimmortalizing B cells from said animal immunized with said antigen,screening the resulting immortalized cells for the secretion of saidimmunoglobulin specific for said antigen, and

-   -   (i) recovering immunoglobulin secreted by said immortalized B        cells, or    -   (ii) recovering the genes encoding at least the immunoglobulin        from the immortalized B cells, and optionally modifying said        genes;    -   (iii) expressing said genes or modified forms thereof to produce        the immunoglobulin or analog; and    -   (iv) recovering said immunoglobulin or analog.

In short, the genes encoding the immunoglobulins produced by thetransgenic animals of the invention can be retrieved and the nucleotidesequences encoding the variable region can be manipulated according toknown techniques to provide a variety of analogs such as those describedabove. In addition, the immunoglobulins themselves containing thevariable regions can be modified using standard coupling techniques toprovide conjugates retaining immunospecific regions.

Thus, immunoglobulin “analogs” refers to the moieties which containthose portions of the antibodies of the invention which retain theirimmunospecificity. These will retain sufficient variable regions toprovide the desired specificity.

As stated above, all of the methods of the invention includeadministering the appropriate antigen to the transgenic animal. Therecovery or production of the antibodies themselves can be achieved invarious ways.

First, and most straightforward, the polyclonal antibodies produced bythe animal and secreted into the bloodstream can be recovered usingknown techniques. Purified forms of these antibodies can, of course, bereadily prepared by standard purification techniques, preferablyincluding affinity chromatography with Protein A, antiimmunoglobulin, orthe antigen itself. In any case, in order to monitor the success ofimmunization, the antibody levels with respect to the antigen in serumwill be monitored using standard techniques such as ELISA, RIA and thelike.

For some applications only the variable regions of the antibodies arerequired, which can be obtained by treating the polyclonal antiserumwith suitable reagents so as to generate Fab′, Fab, or F(ab″)2 portions.Such fragments are sufficient for use, for example, in immunodiagnosticprocedures involving coupling the immunospecific portions ofimmunoglobulins to detecting reagents such as radioisotopes.

Alternatively, immunoglobulins and analogs with desired characteristicscan be generated from immortalized B cells derived from the transgenicanimals used in the method of the invention or from the rearranged genesprovided by these animals in response to immunization. Thus, as analternative to harvesting the antibodies directly from the animal, the Bcells can be obtained, typically from the spleen, but also, if desired,from the peripheral blood lymphocytes or lymph nodes and immortalizedusing any of a variety of techniques, most commonly using the fusionmethods described by Kohler and Milstein Nature 245 (1975), 495. Theresulting hybridomas (or otherwise immortalized B cells) can then becultured as single colonies and screened for secretion of antibodies ofthe desired specificity. After the appropriate hybridomas are selected,the desired antibodies can be recovered, again using conventionaltechniques. They can be prepared in quantity by culturing theimmortalized B cells using conventional methods, either in vitro or invivo to produce ascites fluid. Purification of the resulting monoclonalantibody preparations is less burdensome than in the case of serum sinceeach immortalized colony will secrete only a single type of antibody. Inany event, standard purification techniques to isolate the antibody fromother proteins in the culture medium can be employed.

As an alternative to obtaining immunoglobulins directly from the cultureof immortalized B cells derived from the animal, the immortalized cellscan be used as a source of rearranged heavy chain and light chain locifor subsequent expression and/or genetic manipulation. Rearrangedantibody genes can be reverse transcribed from appropriate mRNAs toproduce cDNA. If desired, the heavy chain constant region can beexchanged for that of a different isotype or eliminated altogether. Thevariable regions can be linked to encode single chain Fv regions.Multiple Fv regions can be linked to confer binding ability to more thanone target or chimeric heavy and light chain combinations can beemployed. Once the genetic material is available, design of analogs asdescribed above which retain their ability to bind the desired target isstraightforward.

Once the appropriate genetic material is obtained and, if desired,modified to encode an analog, the coding sequences, including those thatencode, at a minimum, the variable regions of the heavy and light chain,can be inserted into expression systems contained on vectors which canbe transfected into standard recombinant host cells. A variety of suchhost cells may be used; for efficient processing, however, mammaliancells are preferred. Typical mammalian cell lines useful for thispurpose include CHO cells, 293 cells, or NSO cells. The production ofthe antibody or analog is then undertaken by culturing the modifiedrecombinant host under culture conditions appropriate for the growth ofthe host cells and the expression of the coding sequences. Theantibodies are then recovered from the culture. The expression systemsare preferably designed to include signal peptides so that the resultingantibodies are secreted into the medium; however, intracellularproduction is also possible.

In addition to deliberate design of modified forms of the immunoglobulingenes to produce analogs, advantage can be taken of phage displaytechniques to provide libraries containing a repertoire of antibodieswith varying affinities for the desired antigen. For production of suchrepertoires, it is unnecessary to immortalize the B cells from theimmunized animal; rather, the primary B cells can be used directly as asource of DNA. The mixture of cDNAs obtained from B cells, e.g., derivedfrom spleens, is used to prepare an expression library, for example, aphage display library transfected into E. coli. The resulting cells aretested for immunoreactivity to the desired antigen. Techniques for theidentification of high affinity antibodies from such libraries aredescribed by Griffiths, EMBO J. 13 (1994), 3245-3260; Nissim, ibid,692-698, and Griffiths, ibid, 12, 725-734. Ultimately, clones from thelibrary are identified which produce binding affinities of a desiredmagnitude for the antigen, and the DNA encoding the product responsiblefor such binding is recovered and manipulated for standard recombinantexpression. Phage display libraries may also be constructed usingpreviously manipulated nucleotide sequences and screened in similarfashion. In general, the cDNAs encoding heavy and light chain areindependently supplied or are linked to form Fv analogs for productionin the phage library. The phage library is then screened for theantibodies with highest affinity for the antigen and the geneticmaterial recovered from the appropriate clone.

Further rounds of screening can increase the affinity of the originalantibody isolated. The manipulations described above for recombinantproduction of the antibody or modification to form a desired analog canthen be employed.

There are large numbers of antigens for which antibodies and theiranalogs would be made available by the methods of the invention. Theseinclude, but are not limited to, the following nonlimiting set:leukocyte markers, such as CD2, CD3, CD4, CD5, CD6, CD7, CD8, CD11a,b,c,CD13, CD14, CD18, CD19, CD20, CD22, CD23, CD27 and its ligand, CD28 andits ligands B7.1, B7.2, B7.3, CD29 and its ligand, CD30 and its ligand,CD40 and its ligand gp39, CD44, CD45 and isoforms, Cdw52 (Campathantigen), CD56, CD58, CD69, CD72, CTLA-4, LFA-1 and TCR;histocompatibility antigens, such as MMC class I or II, the Lewis Yantigens, Slex, Sley, Slea, and Selb;

adhesion molecules, including the integrins, such as VLA-1, VLA-2,VLA-3, VLA-4, VLA-5, VLA-6, LFA-1, Mac-1, amp3, and p150,95; and

the selectins, such as L-selectin, E-selectin, and P-selectin and theircounterreceptors VCAM-1, ICAM-1, ICAM-2, and LFA-3;

interleukins, such as IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8,IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, and IL-15;

interleukin receptors, such as IL-1R, IL-2R, IL-3R, IL-4R, IL-5R, IL-6R,IL-7R, IL-8R, IL-9R, IL-10R, IL-11R, IL-12R, IL-13R, IL-14R and IL-15R;

chemokines, such as PF4, RANTES, MIP1a, MCP1, IP10, ENA-78, NAP-2, Groa,Grow, and IL-8;

growth factors, such as TNFalpha, TGFbeta, TSH, VEGF/VPF, PTHrP, EGFfamily, FGF, PDGF family, endothelin, Fibrosin (FsF,), Laminin, andgastrin releasing peptide (GRP);

growth factor receptors, such as TNFalphaR, RGFbetaR, TSHR, VEGFR/VPFR,FGFR, EGFR, PTHrPR, PDGFR family, EPO-R, GCSF-R and other hematopoieticreceptors;

interferon receptors, such as IFNaR, IFNPR, and IFNyR;

Igs and their receptors, such as IGE, FceRI, and FceRII;

tumor antigens, such as her2-neu, mucin, CEA and endosialin;

allergens, such as house dust mite antigen, lol pl (grass) antigens, andurushiol;

viral proteins, such as CMV glycoproteins B, H, and gCIII, HIV-1envelope glycoproteins, RSV envelope glycoproteins, HSV envelopeglycoproteins, EBV envelope glycoproteins, VZV, envelope glycoproteins,HPV envelope glycoproteins, Hepatitis family surface antigens;

toxins, such as pseudomonas endotoxin and osteopontin/uropontin, snakevenom, spider venom, and bee venom;

blood factors, such as complement C3b, complement C5a, complement C5b-9,Rh factor, fibrinogen, fibrin, and myelin associated growth inhibitor;

enzymes, such as cholesterol ester transfer protein, membrane boundmatrix metalloproteases, and glutamic acid decarboxylase (GAD); and

miscellaneous antigens including ganglioside GD3, ganglioside GM2, LMP1,LMP2, eosinophil major basic protein, PTHrp, eosinophil cationicprotein, pANCA, Amadori protein, Type IV collagen, glycated lipids,v-interferon, A7, Pglycoprotein and Fas (AFO-1) and oxidized-LDL.

As mentioned before, the immunoglobulin or its encoding cDNAs may befurther modified. Thus, in a further embodiment the method of thepresent invention comprises any one of the step(s) of producing achimeric antibody, humanized antibody, single-chain antibody,Fab-fragment, bi-specific antibody, fusion antibody, labeled antibody oran analog of any one of those. Corresponding methods are known to theperson skilled in the art and are described, e.g., in Harlow and Lane“Antibodies, A Laboratory Manual”, CSH Press, Cold Spring Harbor, 1988.When derivatives of said antibodies are obtained by the phage displaytechnique, surface plasmon resonance as employed in the BIAcore systemcan be used to increase the efficiency of phage antibodies which bind tothe same epitope as that of any one of the antibodies described herein(Schier, Human Antibodies Hybridomas 7 (1996), 97-105; Malmborg, J.Immunol. Methods 183 (1995), 7-13). The production of chimericantibodies is described, for example, in WO89/09622. Methods for theproduction of humanized antibodies are described in, e.g., EP-A1 0 239400 and WO90/07861. A further source of antibodies to be utilized inaccordance with the present invention are so-called xenogeneicantibodies. The general principle for the production of xenogeneicantibodies such as human antibodies in mice is described in, e.g., WO91/10741, WO 94/02602, WO 96/34096 and WO 96/33735. As discussed above,the antibody of the invention may exist in a variety of forms besidescomplete antibodies; including, for example, Fv, Fab and F(ab)2, as wellas in single chains; see e.g. WO88/09344. The antibodies of the presentinvention or their corresponding immunoglobulin chain(s) can be furthermodified using conventional techniques known in the art, for example, byusing amino acid deletion(s), insertion(s), substitution(s),addition(s), and/or recombination(s) and/or any other modification(s)known in the art either alone or in combination. Methods for introducingsuch modifications in the DNA sequence underlying the amino acidsequence of an immunoglobulin chain are well known to the person skilledin the art; see, e.g., Sambrook, Molecular Cloning A Laboratory Manual,Cold Spring Harbor Laboratory (1989) N.Y. and Ausubel, Current Protocolsin Molecular Biology, Green Publishing Associates and WileyInterscience, N.Y. (1994). Modifications of the antibody of theinvention include chemical and/or enzymatic derivatizations at one ormore constituent amino acid, including side chain modifications,backbone modifications, and N— and C-terminal modifications includingacetylation, hydroxylation, methylation, amidation, and the attachmentof carbohydrate or lipid moieties, cofactors, and the like. Likewise,the present invention encompasses the production of chimeric proteinswhich comprise the described antibody or some fragment thereof at theamino terminus fused to heterologous molecule such as animmunostimulatory ligand at the carboxyl terminus; see, e.g., WO00/30680for corresponding technical details.

For therapeutic applications, the antibodies may be administered in apharmaceutically acceptable dosage form. They may be administered by anymeans that enables the active agent to reach the desired site of action,for example, intravenously as by bolus or by continuous infusion over aperiod of time, by intramuscular, subcutaneous, intraarticular,intrasynovial, intrathecal, oral, topical or inhalation routes. Theantibodies may be administered as a single dose or a series oftreatments. For parenteral administration, the antibodies may beformulated as a solution, suspension, emulsion or lyophilized powder inassociation with a pharmaceutically acceptable parenteral vehicle. Ifthe antibody is suitable for oral administration, the formulation maycontain suitable additives such as, for example, starch, cellulose,silica, various sugars, magnesium carbonate, or calcium phosphate.Suitable vehicles are described in the most recent edition ofRemington's Pharmaceutical Sciences, a standard reference text in thisfield.

For prevention or treatment of disease, the appropriate dosage ofantibody will depend upon known factors such as the pharmacodynamiccharacteristics of the particular antibody, its mode and route ofadministration, the age, weight, and health of the recipient, the typeof condition to be treated and the severity and course of the condition,frequency of treatment, concurrent treatment and the physiologicaleffect desired.

In a still further embodiment, the present invention relates to avaccine comprising a TIRC7 antagonist/inhibitor such as any one of thosedescribed above. This embodiment is based on the surprising finding thatTIRC7 deficient mice exhibit increased T and B cell proliferativeresponse to different stimuli in vitro and in vivo compared with wildtype littermates; see examples 4 to 7. In particular, Type-1 immuneresponse was shown to be pronounced. Accordingly, the invention providesmeans and methods towards the rational design of Th1 adjuvants such asthose discussed in Moingeon, Vaccine 19 (2001), 4363-4372. Methods howto formulate and administer TIRC7 antagonists/inhibitors as vaccinecomponents can be derived from the prior art; see for example Ragupathiet al. in Vaccine 19 (2000), 530-537, which describe the effect ofimmunological adjuvant combinations on the antibody and T-cell responseto vaccination with MUC1-KLH and GD3-KLH conjugates. Hence, in a similarfashion TIRC7 antagonists/inhibitors can be used to augment antibody andT-cell responses against vaccines containing a desired antigen. Anotherexample in this respect is the use of interleukin 12 to enhance thecellular immune response of swine to an inactivated herpesvirus vaccine;see Zuckermann, Adv. Vet. Med. 41 (1999), 447-461. Accordingly, thepresent invention relates to the use of any one of the above describedTIRC7 antagonists/inhibitors as an adjuvant.

This invention will be better understood by reference to the Exampleswhich follow, but those skilled in the art will readily appreciate thatthey are only illustrative of the invention as described more fully inthe claims which follow thereafter. In addition, various documents arecited throughout this application. The disclosures of these documents(including any manufacturer's specifications, instructions, etc.) arehereby incorporated by reference into this application to describe morefully the state of the art to which this invention pertains; however,there is no admission that any document cited is indeed prior art as tothe present invention.

EXAMPLES Example 1 Generation of TIRC7 Deficient Mice

To characterize the functional importance of TIRC7, mice were generatedin which the TIRC7 locus was disrupted by homologous gene targeting(Tivol, Immunity 3 (1995), 541-547). A targeting vector was constructedwhich was used to replace sequences encoding the exons 2-8 of TIRC7 withthe neomycin drug resistance gene (FIG. 1A) (Tivol et al., 1995). Genetargeting was performed in C57 black mice in accordance with establishedprotocols (Capecchi, Science 244 (1989), 1288-1292). In ES cells derivedfrom strain 129 mouse embryos a 2 kb genomic fragment of exon 2 to 8 ofTIRC7 was replaced by insertion of a cassette containing the neomycinresistance gene. Transfection and culturing of the ES cells wasperformed as described by Forster et al., (Cell 87 (1996), 1037-1047).Chimeric males were mated with C57 females and genotype of the offspringwere determined by PCR of tail DNA using oligonucleotide primersdetecting the neomycin cassette, exon 9 and exon 11, in addition toSouthern blotting. Genotyping of the progeny resulting from an intercross of two animals heterozygous for the disrupted TIRC7 gene locus wasperformed as demonstrated by PCR of genomic DNA using TIRC7 specificprimers (FIG. 1B). Homozygous mutant offspring were produced in atypical Mendelian frequency. The lack of TIRC7 protein on CD3 positive Tcells of homozygous offsprings was confirmed by staining with anti-TIRC7antibody and subsequent FACS analysis (FIG. 1C). The phenotype of TIRC7deficient mice demonstrated significantly reduced body weight comparedwith wild type littermates (FIG. 1D).

Example 2 TIRC7 Deficient Mice Exhibits Decrease of All Mononuclear CellPopulations

To examine whether an absence of TIRC7 expression affects development ofcell populations within lymphoid organs flow cytometric analyses wereperformed of single cell suspensions from splenocytes obtained fromTIRC7 deficient and WT littermates (FIG. 1E). Also a significantdecrease of monocytes in mice lacking TIRC7 protein was observed incomparison to WT mice (FIG. 1F).

Single cell suspensions of mouse spleens and lymph nodes were preparedby grinding tissue through a sterile wire mesh and passing through a 50mm filter. All procedures were carried out under sterile conditions inRPMI 1640 medium (Biochom KG) supplemented with 10% fetal calf serum(Biochom KG), 5 mM glutamine, penicillin and streptomycin (Gibco BRL).Cells were stained for 30 mins at 4° C. in 100 μl of PBS and then washedprior to analysis. FACS analysis was performed as described by Waldropet al. (JCI 99 (1997),1739-1750). Cells were stained with a panel offluorochrome-conjugated antibodies, including FITC labeled anti-CD3 mAband anti-CD19 mAb, PE labeled anti-CTLA4, anti-CD3, anti-CD28,anti-CD25, anti-CD69, anti-CD44, anti-CD62L, anti-CD11a, anti-CD71 andanti-CD86 monoclonal antibodies. PerCP labeled anti-B220 and APC labeledanti-CD4 mAb and anti-CD8 mAb antibodies were purchased from PharMingen.Anti-ICOS antibody was purchased from Santa Cruz Biotechnology, anddonkey F(ab′)₂ anti-rabbit as well as goat F(ab′)₂ anti-mouse antibodiesfrom Jackson Laboratories. Analyses were performed by using a FACScanflow cytometer (Becton Dickinson). Cells were analyzed using Cell Questsoftware (Becton Dickinson).

Example 3 Histological Analysis of TIRC7 Deficient Mice Revealed Atrophyof All Immune Tissues

As shown in FIG. 2A, the histological analysis of spleen isolated fromTIRC7(−/−) mice and WT littermates showed a prominent hypoplasia of thesplenic white pulp with disproportioned T and B cell areas. Histologicalmethods have been used essentially as described by Karulin et al. in J.Immunol. 168 (2002), 545-553. Numerous large PALS and small Blymphocytic follicles with a lack of B cell areas were observed inspleens of TIRC7-deficient mice in comparison to WT littermates.Nevertheless, the B lymphocytic follicles of TIRC7 (−/−) spleensrevealed increased numbers of germinal centers. In addition to theregressing white pulp hypoplasia of TIRC7 (−/−) cells, the splenic redpulp revealed a plasma cell hyperplasia, increased numbers of Russelbodies combined with an enhanced granulopoesis which supports thefindings of an increased number of a Gr-1 positive cell fraction,described in FIG. 1F. As shown in FIG. 2B these findings were confirmedby immunohistological staining of the splenic red pulpa whichdemonstrate the hyperplasia of plasma cells in TIRC7 knock out mice.

Histological analysis of the thymus obtained from TIRC7 (−/−) and WTmice revealed different stages of atrophy, predominantly in the cortex.Disintegration and apoptosis of TIRC7 (−/−) thymocytes was strikingcompared to WT littermates. Architectural structure of peripheral lymphnodes of TIRC7 mutant mice lymphocytes in the cortex and paracortex aresparely present. Strikingly, the paracortex of peripheral lymphnodes ofTIRC7 deficient mice exhibit, in contrast to WT littermates an increasednumber of apoptotic lymphocytes, similar to the findings within thesplenic white pulp.

Example 4 Deletion of TIRC7 Leads to Significantly Increase ofProliferative T Cell Response and Induction of Th1 Cytokines in thePresence of Alloantigen or Mitogen

The modulation of TIRC7 signalling with specific antibody inhibitedproliferation of T cells of human PBMC's (Utku et al., 1998). It wastherefore assessed whether TIRC7 deficient mice are able to response tovarious antigens. Cell proliferation assays (assessed by incorporationof [³H] thymidine) were performed in vitro on cells isolated from spleen(FIG. 3A) and lymph node from WT and mutant mice. For T-cellproliferation assays, lymphocytes were stimulated with 10 μg/mlanti-CD3, anti-CD28 mAb (PharMingen) in pre-coated wells or with PHA (1μg/ml or 2 μg/ml)(Sigma) at 37° C. for 48 h in 96-well plates. Cultureswere then pulsed with 0.5 μCi [³H] thymidine (ICN Biochemicals) andafter 18 h incubation cells were harvested and cell proliferation wasdetermined by measuring thymidine incorporation (cpm) using ascintillation counter. As demonstrated in FIG. 3A, splenocytes fromTIRC7 (−/−) mice exhibit a significantly increased proliferativeresponse to anti-CD3 antibody alone, or together with anti-CD28 antibodycompared to WT splenocytes. Similarly, following mitogenic activation ofcells with PHA, a dose-dependent increase in the proliferative responseof cells isolated from TIRC7(−/−) mice was observed, which substantiallyexceeded that observed in cells from wild type animals (FIG. 3A, a andb). 2×10⁶ cell/ml lymphocyte suspensions (from spleen or Iymph node)were incubated with 1.5 μg/ml PHA or 100 ng/ml LPS and 10 ng/ml ofrecombinant IL-4 (PharMingen) at 37° C. for 48 h in microtitre plates.IFN-γ and IL-2 cytokines were measured in supernatants of PHA stimulatedcells. Cytokine levels were determined by ELISA using capture-,detection- and standard-antibodies obtained from PharMingen. PHAactivation of splenocytes from TIRC7(−/−) mice resulted in a significantupregulation of Th1 specific cytokine expression (IL-2 and IFN-γ)compared with that of cells isolated from wild type littermates (FIG.3B).

Example 5 TIRC7 Deletion Affects Expression of Several ActivationMolecules and Costimulatory Molecules on T Cells

Flow cytometric analysis was performed as described in example 2. Theexpression analysis of lymphocyte activation markers CD69 and CD25demonstrated only a moderate increase in resting T cells from TIRC7(−/−)mice compared with control T cells from WT littermates (FIG. 4A).Expression of CD62L and CD44, both representing marker molecules formemory and naive T cell populations, were found to be slightly decreasedand elevated, respectively, compared with wild type cells (FIG. 4A).CD11a staining showed a significant increased memory cell population inT cell from TIRC7 knock out mice in comparison to the wild typelittermates. Resting T cells demonstrated almost threefold more CD11ahigh population (4.9% in knock out and 1.6% in wild type mice) and lowernaïve T cell numbers (17.7%) in comparison with wild type T cellpopulation (28.6%) (FIG. 4B). The effect of TIRC7 deletion oncostimulatory molecules on T cells was examined including CD40L, CTLA4,PD1, CD28 and ICOS as demonstrated in FIG. 4C-D.

CTLA4 molecule is described to be present at higher concentration inintracellular compartments compared to its cell surface expression; seeAlegre, J. Immunol. 157 (1996), 4762-4770. Therefore, CTLA4 expressionanalysis was performed using permeabilized as well as non-permeabilizedlymphocytes for FACS analysis. The intracellular and cell surfaceexpression of CTLA4 was determined by FACS analysis 48 h after mitogenicactivation of T cells. Strikingly, in activated T cells from TIRC7 (−/−)mice only minimal intracellular as well as cell surface expression ofCTLA4 was detectable (FIG. 4C, a-c). In contrast, CTLA4 expression wasunaffected in WT mice and was upregulated after 48 h activation. CD28and ICOS expression levels were significantly decreased on TIRC7 (−/−)cells in contrast to WT littermates (FIG. 4D) whereas no significantchanges in expression was observed for PD1 and CD40L in resting andactivated T cells from TIRC7 mutant and wild type mice.

It is known that a number of cell surface molecules are beingtransported to cell surface via clathrin-coated vesicles. In order toexamine whether TIRC7 deletion affects molecules known to be transportedvia clathrin-coated vesicles to cell surface such as CD71, lymphocyteswere isolated from mutant and WT mice and activated with PHA. Theexpression of CD71 (FIG. 4E) was determined by FACS analysis. Nosignificant differences in the expression of CD71 were observed betweenTIRC7 (−/−) cells and WT mice. These results strongly suggest that TIRC7might deliver distinct signals regulating other signalling pathways ofmolecules known to be essential in immune response.

Example 6 Mice Lacking TIRC7 are More Susceptible to Antigen in vivo

Delayed-type hypersensitivity (DTH) is characteristically mediated by Tcell response and Th-1 cytokines. Targeting of TIRC7 has been shown toaffect the expression of these cytokines; see Utku, Immunity. 9 (1998),509-518. Therefore the functional effects of TIRC7 deletion in mediationof inflammation and leukocyte recruitment was studied during an immuneresponse in vivo by utilizing the DTH reaction. Mice were sensitised toovalbumin by intradermal injection and seven days after immunization,the mice were challenged at day 8 and foot pad swelling was measured.DTH response to ovalbumin (Sigma) was estimated by foot pad swelling aspreviously described in Current Protocols in Immunology. Briefly, micewere sensitised with an injection of 50 μl of 5% (w/v) ovalbumin (ova)emulsified in Freunds Complete Adjuvant (Sigma) at the base of the tail.Eight days after the initial immunization, mice were challenged with aninjection of 30 μl of 2% (w/v) ova in PBS into the left planar foot padand 30 μl of PBS alone in the right planar foot pad. Foot pad thickness(swelling and erythema) were measured in both foot pads and themagnitude of the DTH reaction was determined as the difference in footpad thickness between ova- and PBS-injected foot pads. Foot pad swellingwas peaked 48 h after challenge which was significantly higher in TIRC7knock out mice than observed in wild type littermates (FIG. 5A). Asexpected, assays of Th-1 cytokines revealed even higher levels of IFN-γand IL-2 of TIRC7-deficient mice splenocytes stimulated 48 h withmitogen compared to WT littermates after the ova-challenge. Histologyanalysis of skin obtained form swollen footpads confirmed expectedinflammation signs such as mononuclear infiltration of lymphocytes in WTanimals, which was increased in TIRC7 deficient mice, as shown in FIG.5B.

Example 7 Deletion of TIRC7 Results in Increased B Cell Activation andElevated Immuno Globulin Levels

To further characterize TIRC7 (−/−) role on B cell activation wemeasured cell proliferation of splenocytes in vitro following 48 hincubation with various B cell stimuli including anti-CD40 antibodyalone, or with LPS in combination with IL-4. For B-cell proliferationassays, lymphocytes (2×10⁶ cells/ml) were stimulated with 10 U/ml IL-4and, either 0.5 μg/ml anti-CD40 mAb (Pharmingen) or 0.2 μg/ml LPS(Sigma), for 48 h. Cells were pulsed with 2 μCi [³H] thymidine and cellproliferation was measured after 16 h. Levels of IgM and IgG weremeasured in supernatants of 7 day old cultures by ELISA using capture-,detection- and standard-antibodies obtained from PharMingen, see example2 and 4. As shown in FIG. 6A, in TIRC7 (−/−) splenocytes, in contrast toWT substantially higher levels of proliferation were observed following48 h activation with all B cell stimuli. This was accompanied byincreased levels of IgM and IgG1 production as compared to WT (FIG. 6B).Blood was obtained from the retro-orbital plexus of mice. The serumconcentrations of IgM, IgG1, IgG2a, IgG2b, IgG3, IgA and IgE weredetermined by ELISA using capture-, detection- and standard-antibodiesobtained from PharMingen. The measurement of immunoglobulin levels (Ig)in the serum by ELISA of mutant mice compared to WT showed increasedlevels of all immunoglobulin subclasses supporting the marked B cellactivation in the mutant mice (FIG. 6C).

In order to analyse expression of costimulatory molecules on B cellssplenocytes were incubated in the presence and absence of LPS and IL4and surface expression of CD80 and CD86 was detected by flow cytometry.As shown in FIG. 6D CD86 is upregulated in resting B cells in knock outmice whereas no significant changes in CD80 expression was observed inknock out B cells in comparison to WT littermates, indicating that TIRC7regulates distinct signalling pathways in B cells.

Example 8 Macrophages Revealed Morphological and Functional Defects inTIRC7 Deficient Mice

As shown in FIG. 7A, after 48h of stimulation with LPS and IL-4,TIRC(−/−) peritoneael macrophages showed significantly reduced number ofproliferating cells (KO) compared with wild type (WT). The peritonealcavity was washed with RPMI 1640 medium and the number of macrophageswas determined with Neubauer hemocytometer. 1×10⁶ cells in RPMI 1640medium supplemented with 10% fetal calf serum, 1 mM L-glutamine andstreptomycin-penicillin were stimulated with the LPS (100 ng/ml) andrecombinant IL-4 (10 ng/ml) at 37° C., 5% CO₂ for 48 h. The number ofproliferating cells was quantified by microscopy. The phagocytosisanalyzed by FACS revealed reduction of the overall percentage ofmacrophages and granulocytes showing phagocytosis cells in TIRC7deficient cells compared with wild type. In order to analyze whether theTIRC7 deficiency affects the cytoskeleton which might lead to reducedability of phagocytosis confocal microscopic analysis of TIRC7(−/−)macrophages were performed by using several specific antibodies againstcytoskeleton molecules. Macrophages of the peritoneal cavity were coated1 h at 37° C. on slides pretreated with Poly-L-Lysin (1:10, Sigma) andfixed after 20 min at 4° C. with 4% PFA. Cells were blocked with 5% milkfor 1 h at room temperature and permeabilized with PBS/Triton (100×0.5%) for 10 min at room temperature. Staining was performed usinganti-actin (1:50, Santa Cruz), anti-tubulin (1:50, Santa Cruz) andanti-vinculin (1:50, Santa Cruz) rabbit polyclonal antibodies, or IgGrabbit control antibody (1:50, Santa Cruz) and incubation at 4° C. overnight. The secondary antibody, cy3 labeled anti-rabbit antibody (1:250,Dianova) was incubated for 1 h at room temperature. Staining wasanalyzed using a Pascal 5 confocal microscope. As demonstrated in FIG.7B, TIRC7 deficient macrophages exhibit expression defects off allcytoskeleton proteins tested (actin, tubulin and vinculin).

1. A composition of matter for treating therapeutically orprophylacticly a mammal afflicted with a disorder ameliorated by anincrease in phagocytosis and/or monocyte population, which comprises atherapeutically effective amount of T-cell immune response cDNA 7(TIRC7), an activator of TIRC7 or of a nucleic acid molecule encodingsaid TIRC7 or said activator, and optionally a pharmaceutically orcosmetically acceptable carrier.
 2. A composition of matter for treatingtherapeutically or prophylacticly a mammal afflicted with a disorderameliorated by a decrease in phagocytosis and/or monocyte population,which comprises a therapeutically effective amount of an antagonist ofT-cell immune response cDNA 7 (TIRC7) or of a nucleic acid moleculeencoding said antagonist, and optionally a pharmaceutically orcosmetically acceptable carrier.
 3. The composition of claim 1, whereinTIRC7 is a recombinant TIRC7, a functional derivative thereof or afunctionally equivalent substance.
 4. The composition of claim 1,wherein the composition comprises a stimulatory anti-TIRC7 antibody, aTIRC7 ligand or a cell (over)expressing TIRC7.
 5. The composition ofclaim 2, wherein the antagonist blocks an interaction of TIRC7 and itsligand.
 6. The composition of claim 2, wherein the antagonist is orcomprises an antibody, a (poly)peptide, a nucleic acid molecule, a TIRC7gene targeting vector, a small organic compound, a TIRC7 ligand, peptidenucleic acid (PNA), aptamer, or peptide mimetic.
 7. The composition ofclaim 6, wherein the antagonist is designed to be expressed inmonocytes.
 8. The composition of claim 2, wherein the antagonistcomprises (i) an anti-TIRC7 antibody or an anti-TIRC7-ligand antibody;or (ii) a non-stimulatory form of TIRC7 or of its ligand. 9-10.(canceled)
 11. A method of increasing phagocytosis and/or monocytepopulation, comprising contacting a mammalian cell with an effectiveamount of T-cell immune response cDNA 7 (TIRC7), an activator of TIRC7or of a nucleic acid molecule encoding said TIRC7 or said activator. 12.The method of claim 11, comprising p1 (a) obtaining cells, tissue or anorgan from a subject; (b) introducing into said cells, tissue or organ anucleic acid molecule encoding and capable of expressing TIRC7 or itsligand in vivo; and (c) reintroducing the cells, tissue or organobtained in step (b) into the same subject or a different subject.
 13. Amethod of decreasing phagocytosis and/or monocyte population, comprisingcontacting a mammalian cell with an effective amount of an antagonist ofT-cell immune response cDNA 7 (TIRC7) or of a nucleic acid moleculeencoding said antagonist.
 14. A method of treating therapeutically orprophylacticly a mammal afflicted with a disorder ameliorated by anincrease in phagocytosis and/or monocyte population, which comprisesadministering to the mammal a therapeutically effective amount of T-cellimmune response cDNA 7 (TIRC7), an activator of TIRC7 or of a nucleicacid molecule encoding said TIRC7 or said activator.
 15. A method oftreating therapeutically or prophylacticly a mammal afflicted with adisorder ameliorated by a decrease in phagocytosis and/or monocytepopulation, which comprises administering to the mammal atherapeutically effective amount of an antagonist of T-cell immuneresponse cDNA 7 (TIRC7) or of a nucleic acid molecule encoding saidantagonist.
 16. The method of claim 11, wherein the antagonist oractivator is an agent.
 17. An article of manufacture for administeringto a mammal the composition of matter of claim 1, comprising a soliddelivery vehicle having the composition operably affixed thereto. 18-22.(canceled)
 23. A method to produce an immunoglobulin or an analogthereof, specific for a desired antigen, which method comprises: (a)administering said antigen or an immunogenic portion thereof to anonhuman animal under conditions to stimulate an immune response,whereby said animal produces B cells that secrete immunoglobulinspecific for said antigen; wherein said nonhuman animal is characterizedby being substantially incapable of producing endogenous T-cell immuneresponse cDNA 7 (TIRC7) or TIRC7 activity in lymphocytes; and (b)recovering said immunoglobulin or analog.
 24. A method to produce animmunoglobulin or an analog thereof, specific for a desired antigen,which method comprises administering said antigen or an immunogenicportion thereof to a nonhuman animal under conditions to stimulate animmune response, whereby said animal produces B cells that secreteimmunoglobulin specific for said antigen; wherein the endogenous T-cellimmune response of said nonhuman animal is inhibited by administering anagent as defined in claim
 2. 25. The method of claim 23, wherein theantigen or an immunogenic portion thereof is administered in conjunctionwith an agent.
 26. The method of claim 23, further comprising recoveringsaid polyclonal immunoglobulin or analog from said animal.
 27. Themethod of claim 23, further comprising immortalizing B cells from saidanimal immunized with said antigen, screening the resulting immortalizedcells for the secretion of said immunoglobulin specific for saidantigen, and (i) recovering immunoglobulin secreted by said immortalizedB cells, or (ii) recovering the genes encoding at least theimmunoglobulin from the immortalized B cells, and optionally modifyingsaid genes; (iii)expressing said genes or modified forms thereof toproduce the immunoglobulin or analog; and (iv) recovering saidimmunoglobulin or analog.
 28. The method of claim 23, further comprising(i) recovering genes encoding the immunoglobulins from the primary Bcells of the animal; (ii) generating a library of said genes expressingthe immunoglobulins; (iii)screening the library for an immunoglobulinwith the desired affinity for the antigen; (iv) recovering the genesencoding the immunoglobulin; (v) expressing said genes to produce animmunoglobulin or analog; (vi) recovering said immunoglobulin or analog.29. The method of claim 23, wherein the desired antigen is selected fromthe group consisting of transition state mimics; leukocyte markers;histocompatibility antigens; adhesion molecules; interleukins;interleukin receptors; chemokines; growth factors and their receptors;interferon receptors; Igs and their receptors; tumor antigens;allergens; viral proteins; toxins; blood factors; enzymes; gangliosideGD3, ganglioside GM2, LMP1, LMP2, eosinophil major basic or cationicprotein, pANCA, Amadori protein, Type IV collagen, glycated lipids,γ-interferon, A7, P-glycoprotein, Fas (AFO-1) and oxidized-LDL; humanIL-6 or IL-8, human TNFα, human CD4, human L-selectin, human gp39, humanIgE, human αVβ3, human Fibrinosin (F_(s)F⁻¹), human laminin, humanPTHrP, and tetanus toxin C (TTC). 30-32. (canceled)
 33. The method ofclaim 23, wherein said immunoglobulin or analog is an antibody or analogthereof.
 34. The method of claim 23, further comprising the step(s) ofproducing a chimeric antibody, humanized antibody, single-chainantibody, Fab-fragment, bi-specific antibody, fusion antibody, labelledantibody or an analog of any one of those.
 35. (canceled)
 36. A vaccinecomprising an agent as defined in claim
 2. 37. (canceled)